M2M+Unit+I+LOs

toc //These Learning Objectives were originally compiled by James Rose (C/O 2011)// =Bioenergetics=
 * Define: 1) Entropy, 2) Enthalpy, 3) Free energy, 4) High energy compounds, 5) Oxidation-reduction reaction:
 * Entropy: randomness associated with a given system.
 * Enthalpy: measure of heat content (thermodynamic potential) of a system.
 * Free energy: the amount of thermodynamic energy in a system that can be converted into work at a given temperature and pressure.
 * High energy compounds: compounds with bonds that release greater than 7 kcal/mol of energy when broken (∆G= -7; chemical energy of greater than 7 kcal/mol)
 * Oxidation-reduction reaction: a chemical reaction involving the transfer of electrons away from one or more compounds (the oxidized compounds) to one or more other compounds (the reduced compounds).
 * Review the first and second [|laws of thermodynamics] :
 * 1st law: Energy conservation (cannot be created or destroyed, only converted).
 * 2nd law: The entropy of the universe is constantly increasing. (This means that in any given set of reactions that occur simultaneously in a system, the overall entropy of the system must increase. This usually takes the form of some energy in the system being converted to heat. Does beg the question of where we got so much order to begin with, since the universe is currently progressing towards a state of complete thermodynamic equilibrium (flat, completely uniform and sterile space).)
 * Describe the different forms of energy: (from wikipedia)
 * Kinetic- energy of motion (radiant, thermal, mechanical, electric)
 * Potential- energy stored within a system due to configuration or position, can be converted to kinetic energy to perform work.
 * Radiant- energy of EM waves, as in photons from the sun that power life on earth.
 * Thermal- kinetic energy of molecules (molecular translation, rotation, vibration, e- spin and translation, nuclear spin). Proteins function at an optimal thermal energy.
 * Mechanical- potential and kinetic energy within a mechanical system, discussed within the confines of cellular movement and cellular components.
 * Electric- energy movement of charged particles down gradients of electric potential.
 * Chemical- energy stored in chemical bonds
 * Memorize the following basic thermodynamic equations that involve free energy,equilibrium constant, enthalpy and entropy. Know how to calculate one unknown variable in an equation when all other variables are given.
 * Refers to [|Gibbs Free Energy]
 * a) ∆G = ∆G0 + (RT * ln [PRODUCTS]/[REACTANTS])
 * b) ∆G0 = -RT * ln (Keq)
 * c) ∆G = ∆H – (T * ∆S )
 * Know the relationship between the sign of the standard free energy and the direction of a reaction under standard conditions.
 * ∆G<0 = spontaneous
 * ∆G>0 = not spontaneous
 * ∆G=0 = at equilibrium
 * Describe the effect of positive/negative entropy or enthalpy on the thermodynamic forces driving a reaction based on the equation: ∆G = ∆H – T ∆S.
 * + entropy change increases spontaneity of a reaction (decreases ∆G) (-∆H = exothermic)
 * + enthalpy change decreases spontaneity of a reaction (increases ∆G) (+∆S = increased entropy)
 * Know how to calculate the numerical conversion between free energy (∆G) and Redox potential (∆E) in biological systems.
 * Can calculate ∆E for a reaction by subtracting the Eo of the electron donor from the Eo of the electron acceptor.
 * Eo of a molecule is high when the molecule is more electronegative. It's high negative when the molecule is more electropositive.
 * ∆G = -nF∆E. Notice that when ∆E is positive (so that the electrons are flowing away from an electropositive atom to an electronegative one), ∆G is negative and thus the reaction is more spontaneous. n= # electrons transferred; F= Faraday’s constant; ∆E= difference in reduction potential in volts.
 * Recognize the fact that series of electron transfer in biological system can generate energy (a common way of converting the solar thermal energy into chemical bond energy in plant, or converting the chemical bond energy into redox energy and other forms of energy).
 * (ie. see mitochondrial electron transport chain and oxidative phosphorylation.)
 * Recognize the fact that the standard free energy changes for a set of reactions are additive, or reactions with positive or negative free energy changes can be coupled.
 * Coupling means the SUM of the reaction determines the spontaneity, thus allowing a reaction of +∆G to continue if paired with a -∆G reaction of greater absolute value.
 * Essentially, if you've got two reactions going on at the same time (which is usually what we're talking about when we talk about a "set of reactions"), the ∆G (free energy change) that determines whether or not both of them go forward or not is just the sum of the ∆G of each individual reaction. If the total ∆G is negative, all the reactions will be spontaneous. This is why you can couple a favorable reaction (like ATP->ADP + Pi) to an unfavorable one to make the unfavorable one spontaneous.
 * Know the major high-energy compounds used in biological systems and the principle of energy storage in high-energy compound.


 * Bonds || Compounds ||
 * thioester bonds C-S || acetyl Co-A ||
 * Phosphoanhydride P-O-P || ATP ||
 * P-N || Phosphocreatine ||
 * C-O-P || Phosphoenolpyruvate ||
 * High yield currency (we’re talking Benjamins here) that is easily spent, converted and recharged.

=**DNA/RNA**=
 * Distinguish purines and pyrimidine [|bases], ribose and deoxyribose, ribo- and deoxyribo nucleosides, nucleotides, nucleoside di and triphosphates
 * Purines: adenine, guanine.
 * Pyrimidines: cytosine, thymine, uracil.
 * Ribose is a five-carbon sugar that's the primary building block of ribonucleic acids. Deoxyribose has been de-hydroxylated at the 2' position.
 * Nucleosides are the central ribose sugar and a base attached to it at the 1' position. A nucleoside with one phosphate group attached to the 5' position of the ribose is called either a **nucleotide** or a nucleoside monophosphate. If there's a chain of two phosphates tagged on at the 5' position, it's a nucleoside diphospate; if the chain is three phosphates long, it's a nucleoside triphosphate.
 * Notice that one nucleoside triphosphate, ATP, is the universal energy currency in the body.
 * Evaluate the relative solubility of the different bases and the diseases related to their insolubility.
 * Phosphates are fairly hydrophillic. The bases attached to the ribose are generally more hydrophobic. Among bases, purines are the least soluble of the bunch.
 * Diseases can result from the accumulation of excess purine derivatives in tissues:
 * [|Gout] - build up of uric acid in joints as a precipitant of purines
 * Lesch-Nyhan Disease - Causes severe neurologic symptoms
 * Identify the chemical basis for the 5’ –3’ polarity of DNA and RNA polynucleotide strands DNA and the phosphodiester linkage.
 * Each nucleotide has an open hydroxyl group at the 3' ribose position and a phosphate at the 5' position (as well as a base stuck onto the 1' carbon). It's the binding of one nucleotide's phosphate group to another's OH group that makes the "chain" of DNA/RNA. Thus, at the end of each DNA or RNA chain, there's either a 'spare,' unlinked phosphate group (what's called the 5' end) or a 'spare,' unlinked hydroxyl group (called the 3' end). The phosphodiester linkage is the link between phosphate and hydroxyl.
 * Review the important experiments that helped to establish DNA as the genetic material.
 * Avery, McCloud and McCarty: established DNA as the genetic material with their //Pneumococcus// experiments (Smooth strain killed mice, Rough strain did not. DNA from heat-killed S cultured with R then killed mice).
 * Franklin and Wilkins: x-ray diffraction suggesting a helical structure.
 * Watson and Crick discover definitive double-helical structure.
 * List [|Chargaff’s rules].
 * The molar ratios of total purines and total pyrimidines are roughly equal (G+A=C+T)
 * The molar ratios of adenine to thymine, and guanine to cytosine, are roughly equal. (G=C, A=T)
 * G+C / A+T ratio is different for different organisms
 * Describe the Watson-Crick model for DNA structure, recognize the major and minor grooves the phosphodiester backbone and the base pairs.
 * Briefly: Two strands in a right handed helix. Sugar/phosphate groups on the outside of the helix (makes sense, they're hydrophilic), bases paired and stacked on the inside (also makes sense, they're hydrophobic). Major groove is the larger of the two grooves running down the helix (due to the geometry of the molecules in either side of the helix); minor groove is the smaller. There are about 10 base pairs per turn of the helix. The horizontal distance covered by A-T is almost identical to that covered by G-C.
 * Describe the chemical basis for the stability of the double helix DNA in solution.
 * You'd think all the phosphate groups right next to each other would generate some electrostatic repulsion and destabilize the molecule. But (a) they're mostly neutralized by positively charged species in the cell(magnesium); (b) the base pair linkages give the helix a lot of stability; and (c) adjacent base pairs "stack" on top of each other, providing additional delocalization options for the electrons and giving, often, more stability than comes from the base pairing itself. The stacking interactions are stronger between G/C than A/T.
 * Increased salt concentrations will increase the stability of the DNA molecule (thus increase its melting temperature Tm)
 * Extremes of pH alter ionization of bases which form H-bonds and thus decreases stability
 * Increase in DNA length will increase stability
 * The higher the GC content the more stable the DNA (more H-bonding/delocalization potential).
 * Distinguish between linear and circular and relaxed and supercoiled DNA
 * Linear DNA- large segments cannot distribute twisting and thus also can become supercoiled
 * Circular DNA (prokaryotes)
 * Relaxed DNA- straight ribbon of proper twisting (normal)
 * Supercoiled DNA- DNA under torsional strain that becomes contorted into shapes such as a figure 8, these formations are called writhe -
 * Supercoiling is important for DNA packaging in eukaryotes
 * Describe the chemical modifications of bases in DNA including different forms of DNA damage (methylation, deamination, depurination, UV cross linking) and their significance to disease.
 * Methylation of bases:
 * Methylation tends to occur at the (5') cytosine ends of CpG sequences.
 * This decreases the gene activity, is epigenetically inherited based on hemimethylation.
 * Abnormal methylation leads to abnormal gene regulation
 * Deaminination of bases:
 * If you deaminate cytosine, you get uracil, which shouldn't be in DNA at all- this is usually repaired by base excision repair (see next section).
 * The other, more serious problem is that if the cytosine has already been methylated and it's deaminated, you get thymine-- which is a much subtler change, since you expect to find thymine in DNA anyway. If this isn't corrected before the next replication of the DNA, the mutation can cause one of the replicated helices to contain AT instead of CG at this point.
 * Nitrous acid can greatly speed up deamination (removal of NH)
 * Depurination of bases:
 * Effectively, this is a hydrolysis reaction, breaking off the purine base from the ribose and leaving a hydroxyl group.
 * It's usually caught by DNA repair enzymes.
 * The problem is that it significantly weakens the phosphodiester backbone at the depurination site-- so if you have a couple of these nearby, it can break the backbone.
 * UV-caused cross-linking of bases:
 * Usually occurs between thymines to create [|thymine dimers] . (cyclobutane)
 * Distort the DNA helix and can block replication enzymes.
 * Generally repaired by nucleotide excision repair (see next section) and TFIIH
 * Wear sunscreen.
 * Explain the chemistry of DNA polymerization and how nucleoside analogues are used as drugs.
 * DNA polymerization is covered in the next section. Essentially, you can use molecules that are very similar to nucleosides to block replication of virally infected cells by having the replicating DNA (which recruits free-floating nucleosides as it replicates) incorporate the analogues into the growing chain. The analogues are different enough from actual nucleosides to ensure that the resulting DNA chains are nonfunctional.
 * Notice you can also use differences in the viral reverse transcriptase pathways to design nucleoside analogues preferentially incorporated by reverse transcriptase pathways.
 * More-specific nucleoside analogues usually used against retroviruses. Less specific nucleoside analogues usually used as chemotherapy against cancer.
 * Attacking DNA metabolism can occur through 4 methods
 * Synthesis of precursors (dNTP)
 * Intercalation (getting in the middle)
 * Covalently binding bps
 * Topoisomerases
 * Define the major similarities and differences between DNA and RNA:
 * DNA: has no hydroxyl group at the 2' position of the ribose, which makes it more stable and less prone to hydrolyzation by nucleophilic attack at the 2’ location. DNA binds cytosine, guanine, adenine, and thymine as its bases.
 * RNA: hydroxylated at its 2' ribose position; binds uracil instead of thymine. RNA is usually single-stranded, although it can form double-stranded loops (often called 'hairpin loops') with itself.
 * Define 5 classes of [|RNA] in a human cell.
 * 3 main types: structural, regulatory, and information-containing.
 * Structural
 * **rRNA** (ribosomal RNA)- make up ribosomes and translate **tRNA** (transfer RNA)- move RNA around
 * **snRNA** (small nuclear RNA) and **snoRNA** (small nucleolar RNA) for a variety of in-cell modifications such as splicing.
 * Regulatory
 * **miRNA** (mirco RNA) and **siRNA** (small interfering RNA) to downregulate gene expression.
 * Information-containing
 * **mRNA** (messenger RNA) to be translated into proteins.
 * Describe the chemical basis for nucleic acid melting and annealing and the how it can be used to detect one specific DNA sequence in total cellular DNA.
 * Essentially, you're breaking and re-making the hydrogen bonds between base pairs. A given strand of DNA will re-anneal itself to its complementary pair more or less by itself. This means you can take a bunch of someone's DNA, melt it, throw a tagged DNA strand from something you want to test for in the mix, and see if it anneals to anything. If it does, there's a match in the person's DNA.

=**DNA Replication and Repair**=

** DNA replication and repair **

 * Describe the meaning of "semi-conservative", "bidirectional", "Okazaki fragments", "origin", and "fork" as it relates to DNA replication.
 * Semi-conservative: Each DNA strand is preserved as one half of a new double DNA strand; thus each new DNA strand has one-half original material and one-half newly synthesized material.
 * Bidirectional: Means that when the replication machinery attaches to the DNA double helix, replication proceeds in both directions along that helix at once.
 * Okazaki fragments: Small stretches of DNA synthesized during replication in the 5' to 3' direction (on the lagging strand). Since the synthesized strand is running in the 3' to 5' direction, and since new dNTPs can only be added at the 3' hydroxyl group, the DNA synthesis process takes a kind of "leapfrogging" approach whereby small segments on that strand are copied 5' to 3' and then melded together later.
 * Origin of replication, aka replication origin: Specific sequences for recognition by binding proteins. Usually contain multiple short repeats, as well as an A-T rich streak. There are hundreds per chromosome, 1 in a prokaryote.
 * Replication fork: where the DNA helicases have unwound the double helix. Effectively the H-bonds of the base pairs have been split apart and the two strands are peeled away from each other, thus forming a "fork" in which the replication machinery sits and synthesizes complementary strands.
 * Know the functions of the following proteins during DNA replication: origin binding proteins, helicase, single-strand binding proteins, primase, Pol I, Pol III, processivity clamp, DNA ligase, telomerase, topoisomerase/gyrase, and reverse transcriptase
 * Origin binding proteins: Proteins bind to the origin and become part of the complex, also recruits Pol III.
 * Helicases: enzymes that catalyze the breaking of H-bonds between base pairs and the subsequent 'unwinding' of the helix.
 * Single-strand binding proteins: bind to the melted strands of original DNA to prevent them from re-annealing or getting messed up. More important for Okazaki fragments as they spend more time single stranded (you really don’t want single stranded stuff hanging around).
 * primase: Enzyme that catalyzes the addition of the RNA primer to begin replication.
 * Pol I: DNA polymerase I, "distributive." Versitile enzyme that replaces the RNA primers using 3 different functions: DNA polymerase, 3-5’ exonuclease activity and 5-3’ exonuclease activity. Does not have the sliding clamp so it is slow and distributive.
 * Common to both Pol III and Pol I: "proofreading" activity, or 3' to 5' exonuclease activity. If the wrong dNTP is added during DNA synthesis, the synthesis stops and 'backs up' slightly (in the 3' to 5' direction) and chops off the last nucleotide added.
 * Pol III: DNA polymerase III, "processive." Synthesizes DNA strand from its complement on both leading and lagging strand. High processivity due to a sliding clamp mechanism that holds the polymerase tightly to the DNA. No 5' to 3' exonuclease activity (thus can't be used to remove RNA primers).
 * Processivity clamp: the aforementioned 'sliding clamp' mechanism. Present in Pol III.
 * DNA ligase: enzyme responsible for sealing Okazaki fragments together once the RNA primers have been replaced by Pol I.
 * (Telomere:) sequence at the ends of chromosomes, consisting of a large number of repeating segments. Gets consistently shorter every time the chromosome is replicated, since the RNA primer on the very last O. fragment can't be replaced by Pol I (Pol I needs to have a nearby 3' OH from the next fragment to bind and replace the RNA primer). After a certain point, the telomeres get short enough that the cell becomes unstable and is destroyed.
 * Telomerase: Enzyme responsible for ensuring that the telomeres of chromosomes in certain immortal structures, such as germ cells, never shorten. Effectively, they act as reverse transcriptases, binding to the ends of DNA sequences and adding on some extra dNTPs. The reason this works is that the telomeres have more or less a uniform repetitious sequence.
 * Topoisomerase/gyrase: Enzyme responsible for relieving torsional strain in the DNA helix in the region ahead of the replication fork. It does this by clipping the phosphodiester backbone in selected places and then putting it back together without strain. Topogyrase is specific to prokaryotes.
 * Reverse transcriptase: Enzyme responsible for copying a base sequence INTO DNA (as opposed to out from it), usually from RNA. This can be endogenous (ie. telomerases) or exogenous (ie. retroviruses).
 * Understand how DNA polymerase creates the phosphodiester bond during addition of dNTPs:
 * It breaks off a diphosphate group from the dNTP and uses the energy liberated from that reaction to bind the remaining phosphate group to the hydroxyl group of the previous nucleotide on the chain.
 * Know that DNA polymerase requires an RNA primer.
 * Know that DNA synthesis only occurs in the 5' to 3' direction. (because that way the phosphates are put right next to the sugar and can couple with phosphodiester creation)
 * Know that errors are corrected by proof reading (3' to 5' exonuclease activity).
 * Understand the order of events that occur during, the differences between, and coordination of, DNA synthesis on the lagging and leading strands
 * Leading strand: pretty simple, relatively speaking:
 * **origin binding proteins** bind to origin.
 * DNA melted apart locally by **helicases.**
 * **Topoisomerases** relieve tension ahead of the replication fork.
 * **Pol III** elongates DNA complementary to leading strand.
 * The two strands, one new, one old, are annealed.
 * Lagging strand: similar but a little more complicated since DNA synthesis can only occur in the 5' to 3' direction. Share first 3 steps with leading strand, then:
 * 1) **Primase** attaches RNA primer to lagging strand segment
 * 2) **Pol III** elongates DNA from RNA primer back a short ways, forming an **Okazaki fragment**.
 * 3) RNA primer is removed and replaced with DNA by **Pol I**.
 * 4) Fragments are sealed together with **DNA ligase**.
 * 5) The two strands, one new and one old, are annealed.
 * Describe the relationship between mutation and cancer, both at the mutational level and at the level of heritable defective human DNA repair pathways, giving examples of human DNA repair diseases
 * Three things seem to need to happen to wind up with cancer. One is that a mutation or mismatching event has to occur in a gene of self proliferation. Another is that the repair mechanisms have to either miss it or be overwhelmed by too many such events (ie. exposure to lots and lots of UV radiation). The third is that the self-destruction pathways cannot be activated (mutated). If all three conditions are met, can result in cancer.
 * Couple of examples of diseases resulting from mutations in DNA repair mechanisms: Cockayne's syndrome, Xeroderma pigmentosum (generally involved with light sensitivity, neurodegeneration, premature aging, and cancer).

** DNA Repair **

 * Explain the basic steps of mismatch repair, describing the type of damage repaired by this pathway, and understand the marking of the old strand of DNA by methylation in E. coli
 * Mismatched base recognized soon after synthesis (before methylation) on //new// strand (not old strand; old strand recognized by methylation in //E. coli//, mechanism of recognition unknown in humans). A stretch of DNA behind and in front of the mismatch is clipped by endonucleases, excised by helicase and exonucleases, and replaced with the correct sequence by DNA Pol III (and sealed with liagases).
 * Describe the basic mechanism of base-excision repair, nucleotide-excision repair, recombinational repair, NHEJ and the types of lesions corrected by these pathways:
 * Nucleotide-excision repair (NER): tends to repair more overt modifications that alter the helical pattern of the affected DNA. Process: recognition, clipping the backbone by endonucleases, excision of the affected part, replacing by Pol I, resealing by DNA ligase.
 * Notice that the recognition pathways here need a transcription factor, **TFIIH**, to work properly. (needs the helicase to melt the DNA)
 * Notice also that there's two kinds of NER:
 * __Transcription coupled NER__: the distortion is within a gene being actively transcribed
 * __Global Genome NER__: the distortion isn't within a gene being actively transcribed, goes back over the whole thing.
 * Base-excision repair (BER): tends to repair subtler modifications, like a mismatched base pair not caught by either proofreading or mismatch repair. Process: recognition, clipping off the inappropriate base by glycosylases, clipping the backbone by endonucleases, chewing off by exonucleases of the affected part, replacing by Pol I, resealing by DNA ligase.
 * [|Recombinatorial repair] : also called homologous recombination. Repairs double-stranded breaks or crosslinks in DNA. Process: partially degrades sides of the break to create primers for DNA synthesis. Copies intact, homologous sequence from other chromosome that aligns with it. Each strand aligns itself with a strand on homologue and fills in its gap from that strand.
 * NHEJ [Non-Homologous-End-Joining]: A form of double-stranded break repair that doesn't involve the homologous chromosomes. Essentially you unwind the two ends with helicases, pair up a few matching bases, and reseal the phosphodiester backbone. Note that this can be inaccurate, as you often lose a few bases off the unpaired strands during the resealing. (handout p.12 has a good picture of this.)
 * Describe the sources and nature of lesions to DNA, the type of repair pathway used to repair the lesion and the molecular consequences of failure to repair the lesions, for:
 * thymine dimers: Good candidate for nucleotide excision repair. Usually caused by UV radiation causing linkage between adjacent thymine residues, causing a bulging deformation of the helical structure. If unrepaired, can cause problems with normal processing due to malformed helix. Also will cause Pol III to fall off and Pol II take over, leading lots of errors
 * uracils in DNA: Good candidate for base excision repair. Usually caused by the deamination of a cytosine residue to produce uracil in the DNA. If uncorrected, can cause problems with both the process of replicating/transcribing this portion of the DNA and also with recognition sites of transcription enzymes.
 * Specifically, if this portion of the DNA is transcribed or replicated as is, it will pair with an adenine residue, not a guanine; thus will have effectively swapped a C for a T.
 * bulky chemical adducts: like thymine dimers, except usually caused by chemotoxic binding of large molecules to bases in a DNA helix. (nucleotide excision repair)
 * double-strand breaks: Good candidate for either homologous recombination repair or non-homologous end joining (good to know: non-homologous end joining is the major form of double-strand break repair). Caused by a double break of the phosphodiester backbone (not sure what underlying causes are). If unrepaired, since a chromosome is one long DNA sequence, can lose up to half of the chromosome (very bad).
 * Describe the mechanism that enables replication to continue in the face of DNA lesions, and know the unfortunate consequence of this process for the cell.
 * The mechanism is called lesion bypass polymerization and usually occurs when the cell doesn't have enough resources to fix all the thymine dimers occasioned by UV exposure.
 * Big damage stops DNA Polymerase III, if NER cannot occur then two bypass polymerases bind to the complex. This creates a conformational change in the complex placing the bypass polymerases across from the damage where they add nucleotides without proof reading. After the damage the complex changes conformation and returns to DNAP III doing its business with proof reading.
 * The error rate is 2 to 4 orders of magnitude higher than normal replication, thus frequently results in cancers, etc.

=**Transcription**=
 * Describe the chemical reaction catalyzed by RNA polymerase and why it is unidirectional.
 * RNA polymerase catalyzes phosphodiester bond formation between ribonucleasides in 5’ to 3’ direction, based on template of 3’ to 5’
 * Need triphosphate nucleoside to make hydrolysis spontaneous
 * Catalysis means this reaction is done 1 way (unidirectional)
 * Distinguish five steps in the transcription cycle common to bacterial and eukaryotic RNA polymerases. (Initiation, Elongation, Termination)
 * 1: RNA polymerase binds to promoter sequence on the helical DNA in a "closed complex."
 * 2: Polymerase melts DNA strands apart near transcription start site, forming an "open complex” aka the "transcription bubble."
 * 3: Polymerase catalyzes phosphodiester linkage of two initial rNTPs.
 * 4: Polymerase advances 3' to 5' down template strand, melting DNA and linking rNTPs.
 * 5: At transcription stop site, polymerase releases completed RNA and dissociates from DNA.
 * Note that steps 1-3 are called **initiation**, step 4 is **elongation** , and step 5 is called **termination**.
 * Name the four cellular RNA polymerases and their main functions.
 * //E. coli// RNA polymerase: transcribes all RNA in //E. coli//.
 * [human] RNA polymerase I: makes ribosomal RNA or rRNA.
 * [human] RNA polymerase II: makes messenger RNA (mRNA), small nuclear RNA (snRNA), and microRNA (miRNA). Note that RPol II has a C-terminal domain (CTD).
 * [human] RNA polymerase III: makes primarily tRNA.
 * Define a promoter and name sequence elements characteristic of promoters in human genes.
 * A promoter is a sequence of DNA upstream of the transcription start site that positively affects the expression of the gene. (where RNA Polymerase binds)
 * Consensus elements: -30 (TATA box), initiator, promoter
 * The TATA box is a frequently conserved TATA sequence about 30 bases upstream from the start site. Mutations in the TATA box often result in reduced expression of the gene (ie beta-thalassemia with B-hemoglobin), because the TATA box binding protein (TBP) helps in assembly of the pre-initiation complex of general transcription factors at the promoter.
 * Initiator is +1 in some but not all eukaryotic genes
 * Promoter proximal elements are promoter DNA sequences between 30-1000 bp upstream of the start site.
 * Enhancer elements are promoter DNA sequences much farther upstream (10,000-50,000 bp). This acts through DNA looping.
 * Describe how alpha-amanitin and rifampicin block transcription.
 * alpha-amanitin: extremely toxic substance found in death cap mushrooms. Acts by inhibiting the movement of RNA Pol II, binding its bridge substructure so that translocation of the polymerase down the DNA chain can't happen.
 * rifampicin: broad-spectrum antibiotic. Acts by binding the beta subunit of bacterial RNA polymerase, plugging up the exit chamber where assembled RNA exits the transcriptional complex. Thus elongation is prevented from going farther than a few base pairs due to having nowhere to go.
 * Name 4 components of the RNA polymerase II pre-initiation complex.
 * TFII [transcription factor II] A, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
 * Of these, TFIID and TFIIH are of particular interest:
 * TFIID (TBP) binds the TATA box on the DNA sequence.
 * TFIIH facilitates nucleotide excision repair, adds PO4 to C-terminal domain of Pol II, and act as helicases to open DNA strands.
 * Describe the clinical syndromes caused by mutations in TFIIH subunits.
 * Problems with nucleotide excision repair: Cockayne's syndrome, Trichothiodystrophy, **Xeroderma pigmentosum** - light sensitivity, abnormal pigmentation, cancer susceptibility, neuorological abnormalities, unscheduled DNA synthesis).
 * Describe the three major ways in which most pre-mRNA's are processed.
 * Capping: replacement of the 5' triphosphate (from the first rNTP to be added) with a backwards, 7-methylguanosine (no phosphate group).
 * Splicing: excision of introns and desegmentation of exons.
 * Cleavage/Polyadenylation: cleavage of RNA at 3' end past the **consensus sequence** and polyadenylation (> 200 A's) of cleaved site.
 * Note that these processing steps take place __while the RNA is still being made__, not after it's finished and released. For example, the cleavage/polyadenylation step is partly responsible for Pol II being released from the DNA.
 * Compare and contrast a pre-mRNA with a mature mRNA.
 * Pre-mRNA: considerably longer (introns are longer than exons), tri-phopshate group at 5' end not cap, no poly-A tail at 3' end.
 * List the functions of the 5' cap of the mRNA.
 * It makes the 5' end resistant to exonucleases (which target the "lone ends" of single D/RNA strands).
 * It helps with splicing and processing through a **cap-binding complex** that recognizes the cap. (primes for splicing, 3’ tail, translation)
 * Translation factor eIF4E (eukaryotic initiation factor 4E) recognizes the cap for transport to the ribosomes.
 * When the cap is eventually removed, it signals for the mRNA to be degraded.
 * List the three reactions required to add a 5' cap to pre-mRNA.
 * (1) Cut off the last PO4 from the triphosphate group at the 5' end of the mRNA.
 * (2) Add guanosine triphosphate (GTP) backwards via guanylyl transferase. It loses two PO4 groups of its own (making GMP) and forms a 5'-to-5' triphosphate bond (2 PO4 from mRNA, 1 PO4 from GMP) to the end of the mRNA.
 * (3) Methylate the 7-position of the guanosine cap via //S// -adenosyl methionine (SAM; nearly universal methyl donor in cell; donates methyl to form homocysteine) to form 7-methyl-guanosine at cap.
 * Memorize the conserved sequences at the 5' and 3' ends of most introns and the consensus sequence at the polyA site.
 * Splice site at 5' end of intron: **GU**.
 * Splice site at 3' end of intron: **AG**.
 * Consensus sequence at poly-A site: **AAUAAA**.
 * Describe how alternative splicing permits multiple proteins to be produced by splicing defects.
 * (normal) Retain or remove exons, mutually exclusive exons, exon truncation.extension at 5’ end, exon truncation/extension at 3’ end, intron retained or removed.
 * Since the 5' splice site (the one identified by snRNA that recruits the spliceosome) at any given intron is part of the DNA sequence, it's vulnerable to being corrupted by mutation and being unrecognizable as an intron, in which case the finished mRNA has an extra sequence in it and the final protein winds up significantly different.
 * Provide examples of genetic disorders caused by splicing defects.
 * Marfan's syndrome: caused by mutations that disrupt splicing of the fibrillin gene transcript (fibrillin is a connective tissue protein that is important for the integrity of the walls of the heart and blood vessels). They are tall and prone to aneurysms.
 * Abnormal splicing of CD44 (cell-surface glycoprotein) is a predictor of tumor metastasis. Used as diagnostic and prognostic marker.
 * Describe the function of U1 and U2 snRNA's in splicing.
 * U1snRNA binds to the GU 5' splice site of introns.
 * U2snRNA binds to the branch point (A) on the pre-mRNA sequence between the 5' and 3' splice sites
 * U2AFsnRNA binds to the 3’ splice site of intron (AG)
 * Lariat splicing mechanism: the U1snRNP brings the 5' splice site into proximity to the branch point. The U2snRNP activates the 2' hydroxyl group at the branch point, which attacks the phosphodiester bond just past the GU 5' splice site. Now there's a free 3' OH on the end of the 5' exon, which attacks the phosphodiester bond at the AG 3' splice site-- linking the two exons and excising the snRNP/intron complex to be degraded.
 * Identify on a diagram of a gene the following:
 * a) transcription start site: Look for either the +1 position or the little bent arrow coming out of the sequence at this point.
 * b) introns: Should be between the GU (5') and AG (3') splice sites.
 * c) 5' splice sites: Look for GU.
 * d) 3' splice sites: Look for AG.
 * e) branch points: should be in the introns, between the GU (5') and AG (3').
 * f) exons: should be between the introns. Mark the introns with GU at 5' and AG at 3', then look between them for the exons.
 * g) 5' UTR: look for a region between position +1 (start of transcript) and the start codon (see below) on the processed mRNA strand.
 * h) 3' UTR: look for a region after the stop codon (see below) until the end of the transcript, including the consensus sequence and the poly-A tail.
 * i) initiation codon: also called start codons. Encodes methionine. Look for 5' AUG.
 * j) termination codon: also called stop codons. Look for 3' UAG, UAA, or UGA.
 * k) poly A site: look for consensus sequence AAUAAA
 * Describe the two reactions that make the mature 3' end of mRNA's.
 * (1) recognition of consensus sequence at pre-mRNA's 3' end (AAUAAA) and cleavage of the mRNA soon after this sequence.
 * (2) polyadenylation of free hydroxyl at 3' end. (not coded for)
 * Describe the relationship between 3' end processing of the pre-mRNA and termination of transcription at the end of a gene.
 * The cleavage and polyadenylation occur while the RNA polymerase continues, it could soon fall off or it could continue to make another mRNA.
 * The poly-A tail seems to be necessary for the RNA polymerase complex to detach from the DNA (termination).
 * Describe the two major functions of the mRNA's poly A tail.
 * protection from degradation
 * export of mRNA from nucleus
 * Provide an example of how alternative poly A sites can be used to make more than one protein from a single gene.
 * two different forms of immunoglobulin M (IgM), membrane-bound and secreted, are formed by alternative poly-A sites in their common gene. This can occur because transcription continues past poly-A site depending on which one is used it will make 2 variants (heavy and light chains).

=**Gene expression**= (10/22/07)
 * List the different eukaryotic DNA control elements:
 * Notice that "DNA control elements" mean transcription-influencing segments of DNA on or associated with the gene being transcribed.
 * 1) TATA box/initiator sequence: usually 25-35 bp upstream from start site. Determines site of transcription initiation and directs binding of RNA Pol II. Also the site at which GTPs (general transcription factors, like TFIID and TFIIH) bind.
 * 2) promoter proximal element: usually within 200 bp upstream of start site, about 20 bp long. Bound by transcription factors to regulate transcription.
 * 3) enhancers: usually much farther upstream, or downstream, than promoters, although still fairly short in themselves (8-20 bp). Can be upstream of the start site, downstream of the last exon, or within introns in the gene itself. Similar function to promoters.
 * Describe a disease that arises from a mutation in a DNA control element, and how the mutation leads to the disease state.
 * Beta-thalassemia (from //thalassa//, Greek for sea): mild inherited anemia (low hemoglobin count). Caused, here, by a mutation in the promoter of the b-globin gene, resulting in lowered rate of production of b-globin protein. (less promotion = less transcription)
 * Gamma-delta-beta thalassemia: more serious anemia caused by a deletion in the locus control region for the transcription of all globin genes, resulting in the loss of globin transcription.
 * Hemophilia B Leyden: X-linked disease (usually males) that affects clotting. Again, a problem in the promoter region of a clotting protein gene. Tends to get partially better at puberty.
 * Fragile-X syndrome: Again, usually a disease of men. Results in mental retardation and atypical development of the face with enlarged testicles (macroorchidism). Caused by an expansion in the CGG count upstream of a particular gene (the FMR1 gene), which results in an abnormally high rate of methylation in that region and transcriptional silencing of the gene.
 * Describe the role of transcriptional activators and repressors.
 * They bind to either the DNA control elements (ie, to the DNA itself) or to other factors bound to control elements (ie, to other proteins that are bound to the DNA).
 * They increase or decrease the rate of transcription of the gene's protein(s).
 * List the two classes of activators and repressors.
 * Those that bind to the control elements in the DNA are called **sequence-specific DNA binding proteins** or SSDBPs. They usually bind to short (6-8 bp) sequences by inserting their alpha-helices into the major groove of the sequence in question.
 * Those that bind to SSDBPs are called **co-factors** . These can both increase or decrease efficacy of transcription factors.
 * Describe the domains of a sequence specific DNA binding protein.
 * Generally two domains. Notice that these domains are modular-- that is, if you pull one domain off of one protein and attach it to another, the second protein will have the function of the domain you just attached.
 * First domain: **DNA binding domain** . Very highly structured and evolutionarily conserved. Binds to the DNA target.
 * Second domain: **activation (or repression) domain** . Fairly unstructured and less conserved; they recruit other proteins (either co-factors or general transcription factors) to bind and affect transcription.
 * Note the difference between co-factors and general transcription factors: co-factors influence the __rate__ of transcription, while GTFs provide the pre-initiation complex needed to __begin__ transcription.
 * List the four major families of sequence specific DNA binding proteins and describe the means for categorizing the proteins into these families.
 * These are categorized based on tertiary structure differences:
 * **Homeodomain proteins** have a helix-turn-helix structure. They tend to be regulators of development and affect many genes at once.
 * **Zinc-finger proteins** have a "finger" made up of two antiparallel beta sheets and an alpha helix, held together by a zinc ion. This finger is what binds with the DNA. The largest family of SSDBPs. Include androgen and estrogen (nuclear hormone) receptors.
 * **Basic leucine zipper proteins (bZIP)** : (the "basic" here refers to the fact that they have a high-pH region that binds to the DNA) Chop sticks. Hydrophobic residue every 7 amino residues, (like leucine). Dimerizes to bind DNA. (c-fos and c-jun).
 * **Basic helix-loop-helix proteins (bHLH)** : also has basic region for DNA binding. Muscle group (MyoD, myogenin).
 * Describe a particular human disorder that arises from a mutation in a sequence specific DNA binding protein, explaining how the mutation leads to the disorder.
 * Craniosynostosis: premature closure of the skull sutures in infants. Arises from a mutation in the homeodomain protein that causes the protein to bind more strongly, creating a "hypermorphic allele" that activates genes more strongly than it should. (upregulates proteins that close sutures via homeodomain mutation)
 * Androgen insensitivity syndrome: Feminization or undermasculinization. Indifference of androgen receptors to androgen hormones. Caused by a mutation in the zinc-finger androgen receptor binding domain or ligand binding domain. This downregulates the transcription of genes controlled by male androgens.
 * Waardenburg syndrome: deafness, pigmentation defects. Associated with mutations in the MITF (microphthalmia-associated transcription factor) gene (which codes for a bHLH binding protein that regulates melanocyte development).
 * Describe combinatorial control as a mechanism for controlling gene expression.
 * Combinatorial control refers to the fact that SSDBPs can dimerize, making possible a wide variety of possible DNA binding sequences.
 * Note that this is always within classes of SSDBPs (e.g. zinc-finger with zinc-finger)
 * For example:
 * SSDBP can dimerize into homodimers and heterdimers, the combination of these proteins leads to many different transcription factors. If you have 4 monomers that can mix and match into dimers you have 2^4 transcription factors. Larger complexes, trimers and tetramers, are also possible.
 * With 1,000s of TF genes you can have 10,000s of TFs (Not 2^1000 - not all TFs can form heterodimers with each other).
 * List the 2 classes of chromatin remodeling factors and briefly describe how they work.
 * DNA-dependent ATPases: these disrupt the histone octamers, opening up the chromatin and exposing DNA for binding via hydrolysis. (SWI/SNF complex)
 * Factors that reversibly modify histones through acetylation of the N-termini:
 * Acetylators, co-activors (**histone acetyltransferases, or HATs** )
 * De-acetylators, co-repressors (**histone deacetylatases, or HDACs** ).
 * Acetylation is associated with increased transcription of the DNA on the affected histone; deacetylation is associated with the reverse.
 * Current theory is that HATs' pattern of histone acetylation recruits co-factors to effect increased transcription rather than directly affecting it themselves by opening up the histone complexes.
 * Define HATs and HDACs and describe how their activity influences transcription.
 * See above.
 * Give an example of a disease in which histone acetylation is altered, and describe the defect that leads to altered histone acetylation.
 * Leukemia: haematopoietic (formation of blood cell components) disease involving chromosomal translocations over-activating fusion proteins that alter the activity of HATs or HDACs.
 * [|Rubinstein-Taybi syndrome] : growth and mental retardation, broad thumbs and toes, craniofacial dysmorphism. Results from mutations in one copy of CREB binding protein gene (haploinsufficiency); CBP is a widespread HAT important in development and its insufficiency has particularly drastic effects.
 * Describe how activators/repressors modulate transcription via their interaction with general transcriptional machinery vs. with chromatin.
 * Activators/Repressors can either:
 * (1) bind to general TFs or the RNA Pol II complex to influence initiation or elongation of the primary transcript
 * (2) interact with chromatin to regulate the accessibility of the DNA to the Pol II transcription apparatus via acetylation, phsophorylation, methylation, or ubiquitination.
 * Discuss the basic principles of transcriptional regulation including how specificity is achieved and how protein-DNA interactions contribute to transcriptional control.
 * (1) Specificity depends on binding of transcriptional activators/repressors to DNA control elements. (binds to specific section of DNA)
 * (2) Regulation depends on DNA-protein and protein-protein interactions. (activation, repression)
 * (3) The interactions affect the conformation of DNA, modification of chromatin structure, and formation of the transcription initiation complex. (change access to gene)
 * (4) Control elements are combinatorial, which allows for thousands of transcriptional activators/repressors to alter the expression of various genes in response to varying stimuli. (mix and match monomers)
 * List at least 4 mechanisms by which sequence specific DNA binding proteins are regulated.
 * (1) Alter the conformation of the TF with ligand binding
 * (2) Regulate entry into the nucleus (regulate access to DNA)
 * (3) Regulate amount of TF in the cell
 * (4) Regulate DNA binding action of protein
 * (5) Phosphorylation/dephosp of TF (affecting degradation, recruitment of of co-activators, DNA binding)
 * Describe how the activity of nuclear hormone receptors is controlled, and how [|Tamoxifen] acts in breast cancer therapy.
 * Steroid hormone enters cell and binds to the nuclear hormone receptor, leading to conformational change, recruitment of coactivators/repressors, and entry into the nucleus. They have zinc-finger DNA binding motif
 * Tamoxifen acts as an antagonist to estrogen, binding to the estrogen receptors as a ligand without providing dimerization-- thus effectively preventing estrogen from binding to its site of action and preventing the transcriptional effect of estrogen receptors.
 * Give an example of a sequence specific DNA binding protein regulated by nuclear entry and describe the mechanism by which its entry is controlled.
 * NF-κB: normally bound to IκB hiding the NLS, which holds it in the cytoplasm (and away from genetic material). Under certain conditions, IκB is phosphorylated, which targets it for degradation. Degrading IκB shows the NLS of NF-κB to migrate into the nucleus and affect transcription. (Asprin blocks phosphorylation of IκB and acts as an anti-inflammative and one of the reasons low-dose aspirin is given to prevent buildup of atherosclerotic plaque).
 * Describe how the amount of an activator/repressor can be regulated within the cell.
 * Specific genes can target activators or repressors for degradation (like APC targets beta-catenin, a cell proliferation activator protein). Lack of Wnt signaling leads to phosphorylation and thus degradation of beta-catenin via the ubiquitin pathway.
 * Clinical fact: ~50% of colon polyps observed in the clinic are caused by mutations in the APC gene, resulting in insufficient degradation of (and thus proliferation of) B-catenin. Not enough APC to phosphorylate the beta-catenin and degrade it to prevent it from getting into the nucleus.
 * Describe a mechanism by which the DNA binding activity of a sequence specific DNA binding protein can be inhibited.
 * Id proteins have a helix-loop-helix (HLH) domain, but no basic domain-- recall that the basic domain is what allows HLH proteins to bind to DNA, while the HLH domain allows dimerization of these proteins. Recall also that bHLH proteins usually dimerize to be able to bind to DNA.
 * When an Id protein heterodimerizes to a good one then it doesn’t bind well to the DNA and thus decreasing the effectiveness of the HLH proteins present. (diluting effectiveness).Also faster and more energy efficiency.
 * List a protein modification that can alter the activity of a sequence specific DNA binding protein, and explain the mechanism by which the activity is altered.
 * Ligand binds G-protein, phosphorylates CREB protein, recruits CBP (CREB binding protein – a HAT) which recruit RNA Pol II leading to transcription of the gene.
 * No phosphorylation, no CREB mediated transcription.
 * Aside from transcriptional regulation, list at least 3 additional mechanisms to control levels of gene expression.
 * Control of mRNA export from nucleus (cap and poly-A tail)
 * Control of mRNA degradation (ie small interfering RNA action)
 * Control of efficiency of translation (ie next lecture IRE/IRP at 5' UTR)
 * Control of protein degradation (ie ubiquitination)

=**Translation**=
 * Understand the steps in the initiation of protein synthesis in prokaryotes and eukaryotes and the differences between prokaryotic and eukaryotic translation initiation:
 * Prokaryotic initiation of translation: dependent on a specific RNA sequence called the ** [|Shine-Dalgarno sequence] ** (8-13 bp) that is upstream from the start codon (AUG). S-D sequence binds to 30S subunit of the prokaryotic ribosome. There can be multiple S-D sequences per mRNA transcript, allowing one transcript to code for multiple proteins (ie **polycistronic** ).
 * Eukaryotic initiation is complex to allow for regulation. It relies on multiple initiation factors assembling a translation complex at the Kozak sequence to position the ribosome at the start codon (eIF = eukaryotic initiation factor):
 * eIF-4E (cap-binding protein) binds to 5' cap.*
 * eIF-4G (scaffolding protein) is recruited by eIF-4E.*
 * eIF-3, which binds to the 40S ribosome.
 * eIF-2, which binds to a tRNA charged with methionine initiates protein synthesis*
 * eIF-4A, a helicase to melt any RNA structure encountered.
 * After the pre-initiation complex (40S + eIFs) becomes the initiation complex (adds cap-binding complex and attaches to RNA), it "scans" down the mRNA until it finds the start codon. Then the eIFs break off and the 60S joins to form the full ribosome (80S).
 * Notice that the 5' UTR can influence the rate of this process or can stop it entirely, depending on how convoluted it is (remember that the ribosome complex has to scan through the 5' UTR to get to the AUG start codon). This is the cap/scanning model.
 * Notice that the start codon, AUG, means that the first amino acid on the translation is Met.
 * Understand the mechanism by which eukaryotic cap-dependent translation is inhibited by certain viruses and how these viruses initiate translation:
 * Certain viruses (rhinovirus, poliovirus, etc) prevent eIF-4E and eIF-4G from coming together and forming the initiator complex.
 * Instead of participating in cap-dependant translation, ribosomes are free to bind to areas of the viral 5' UTR called **Internal Ribosomal Entry Sites** ( **IRESes** ) (as well as any host IRESes present). Thus predominantly the viral mRNA will be translated instead of the host mRNA.
 * Understand the general characteristics of the genetic code, including the start and stop codons:
 * mRNA structure: 5' cap, 5' UTR, start codon (AUG), open reading frames, stop codon (UAG, UAA, or UGA), 3' UTR, poly-A tail.
 * Understand the role of tRNA, the process of charging tRNA and the concept of "humanizing" a protein, and the wobble rule:
 * tRNA: brings in amino acid residues to be added to the polypeptide chain. Consists of acceptor stem where amino acid attaches, the anticodon stem and anticodon loop.
 * Amino acid residues are "activated" by adenylation prior to being attached to a tRNA by aminoacyl tRNA synthetase. Once a tRNA has a residue attached to its acceptor stem it is "charged" and can be used to transfer that residue onto the polypeptide chain.
 * "Humanizing" a protein: Isoacceptor tRNAs are found in varying concentrations in different organisms. To make a human gene easily translatable by a bacterium the genetic code should be changed such that the appropriate codon/tRNA pairings are used while conserving the primary structure (amino acid sequence).
 * The wobble rule: the third base in the codon can vary while still conserving the amino acid coded for (be recognized by the same tRNA).
 * Rule 1: The 5' base of the anticodon always pairs with its expected base partner (A=U, C=G).
 * Rule 2: For the 5' anticodon base or 3' codon base, inosine will pair with any base but G.
 * Rule 3: If the 5' base of the anticodon is G or U, it will bind an additional U or G, respectively. As a tribue to Anand, we'll call this "interpromiscuity."
 * Example 1: If the 5' base of the anticodon is U:
 * 1. U would be expected to bind A.
 * 2. U will bind inosine.
 * 3. U will bind G.
 * Therefore, a U base in the 5' anticodon position can bind A, G or inosine.
 * Example 2: If the 5' base of the anticodon is G:
 * 1. G would be expected to bind C.
 * 2. G will not bind inosine.
 * 3. G will bind U.
 * <span style="border-collapse: collapse; font-family: arial,sans-serif; line-height: normal;">Therefore, a G base in the 5' anticodon position can bind C or U.
 * Understand the mechanism of peptide bond formation and the processes of elongation and termination:
 * Peptide bonds are formed by nucleophilic attack of the alpha amino nitrogen in the A site on the alpha carboxyl/carbonyl carbon in the P site. This is catalyzed by peptidyl transferase (part of ribosome). The energy used to charge the tRNA is used to form the peptide bond.
 * Elongation:
 * 3 sites on ribosomes: A (aminoacyl site), P (peptidyl site), E (exit site).
 * t-RNAs recruited to the A site
 * t-RMA-Methionine enters A, moves to P
 * The next tRNA comes into the A site and with GTP hydrolysis undergoes a conformational change placing the A site residue next to the P site residue
 * The Met residue hydrolyzed to the residue on the A site, catalyzed by peptidyl transferase using energy from ATP used in t-RNA charging (might use the PPi but don’t know details)
 * The "uncharged" tRNA moves to E site where it can be dislodged.
 * The polypeptide-bound tRNA moves from the A to the P site and another charged tRNA comes in at the A site.
 * Repeat. Repeat. Repeat.
 * Termination: ribosome reads a stop codon (UGA, UAG, UAA). A release factor binds to the stop codon cleaving the final t-RNA in the P site from the peptide chain.
 * Understand the mechanisms by which antibiotics inhibit protein synthesis:
 * Antibiotics inhibit translation by interfering with the ribosome (both 30S and 50S subunits) through tRNA binding, elongation and peptidyl transferase.
 * The cancer treatment [|Rapamycin] phosphorylates 4E-BP so that the initiation complex cannot be formed, downregulating translation.
 * Know the various kinds of mutations that affect translation:
 * Point mutations, or changes in a single base pair:
 * **nonsense mutations** : Result in a stop codon where there should be an AA-coding codon. This creates truncated proteins, either nonfunctional or dysfunctional, that are usually quickly degraded by the cell.
 * **missense mutations** : Result in a different AA from the original being encoded by mRNA. Can create a protein with different folding and binding characteristics, or may not be a big deal.
 * **silent mutations** : Result in no change at the protein level. Eg. a mutation from one codon coding for tyrosine to another codon coding for tyrosine.
 * **Read through, reverse terminator, sense mutation** : deletes a stop codon.
 * Deletion or insertion mutations:
 * Major problem here is a **frame shift** . If you're reading codons three at a time, and one base pair gets deleted, it throws off the reading frame (correct "decoding") of the entire gene after that point. This pretty much FUBARs your protein.
 * Note that if you deleted three base pairs at once, you would have no frame shift but would lose an amino acid residue from the final protein.
 * RNA code can be altered after it is made, such as to create a stop codon early in the apolipoprotein B. Deamination creates a truncated protein that is isolated to the intestine while the full-length protein is in the liver.
 * Understand how fluctuating intracellular iron concentrations can alter translation of specific mRNAs:
 * Iron regulation is critical to proper biological functioning, high levels are toxic. There are two mechanisms to control iron levels, both working through Iron Response Biding Proteins 1 & 2. Low iron means little will bind to IRE-BP allowing it to bind to the Iron Response Element. This stops Ferritin production and stablizes Transferrin receptor mRNA.High iron environments mean iron will bind to IRE-BP preventing it from binding to the IRE, thus ferritin mRNA is translated and TFR mRNA is degraded.
 * Ferritin **sequesters** iron (thus lowering the level of free iron in the cell)
 * Transferrin receptor transports iron **into** the cell.
 * Understand how [|interferon] inhibits viral protein synthesis:
 * Interferon activates two pathways in cells near the one under viral attack:
 * One synthesizes an enzyme (2-5A synthase) that activates an endonuclease which cleaves viral mRNA, preventing translation.
 * The other phosphorylates eIF-2 via eIF-2 kinase, inactivating it. This prevents t-RNA-Met from being recruited to the ribsome and thus viral protein synthesis cannot be initiated.

=**Amino Acids, Peptides and Proteins**=

** Lecture I **

 * Identify alpha carbon, the NH2, COOH, and side chains (R groups) of an [|amino acid].
 * alpha carbon sits between the NH2 and COOH. The alpha carbon also sits between a H and the R group/side chain.
 * Distinguish between [|amino acids] with hydrophobic, polar, acidic and basic side chains.
 * Groups discussed in class:
 * Nonpolar/Aliphatic R groups: MAGLIV (great lecturers value my awesome intellect) (methionine, alanine, glycine, leucine, isoleucine, valine) These are not very reactive, only hydrocarbon R groups (+methionine).
 * Aromatic R groups: FYW (phenylalanine, tyrosine, tryptophan) These are mostly hydrophobic and have a ring or two.
 * Polar uncharged groups: SCTPQN (serine, cysteine, threonine, proline, asparagine, glutamine) These have hydroxyl, carboxamide and sulfur groups where electronegativities are unbalanced (+proline).
 * Polar positively charged groups: HRK (histidine, arginine, lysine) These are basic with charged amines (+histidine).
 * Polar negatively charged groups: DE (aspartate, glutamate) These are acidic with resonant oxygens.
 * Understand the function of disulphide bonds within proteins.
 * S-S bonds (formed from two proximal cysteine residues) stabilize both intra-protein (tertiary) and inter-protein (quaternary) structure.
 * Examples are insulin (A and B), keratin in hair and ribonuclease.
 * Describe the major post-translation covalent modifications of amino acid side chains in proteins. Identify the post-translational modification targeted by the disease process or medicine discussed in class.
 * Hydroxylation
 * Hydroxylation of proline residues in collagen stabilizes the structures, mediated by vitamin C. Scurvy is caused by lack of vitamin C and thus underhydroxylation of collagen and is thus weak. Ergo Limey.
 * Carboxylation
 * Carboxylation of glutamate residues on prothrombin, mediated by vitamin K, is required for effective blood clotting. Vitamin K insufficiency leads to improper clotting.
 * **Warfarin** prevents carboxylation of glutamate residues and acts as an anticoagulant.
 * Glycosylation
 * Glycosylation of asparagine residues of proteins on cell membranes and that are secreted increases hydrophilicity.
 * Congenital Disorder of Glycosylation (CDG) has malfunctions in this mechanism.
 * Acetylation/methylation
 * Acetylation and de-acetylation of histones in gene regulation (HDACs and HATS)
 * Cancer treatment can involve blocking HDACs
 * Phosphorylation
 * Reversible phosphorylation via kinases (adds P) and phosphatases (removes P) affects signal transduction
 * Gleevec (tyrosine kinase inhibitor) is a cancer treatment that competitively inhibits bcr-abl kinase such that the substrate is not phosphorylated and active and the tumor cell cannot proliferate via an abberant bcr-able gene.
 * Ubiquitination
 * Ubiquitin added to proteins signals them to be sent to the proteosome for destruction
 * Velcade inhibits a proteosome that degrades good proteins resulting in multiple myeloma.

** Lecture II **

 * Distinguish three covalent bonds including the [|peptide bonds] that make the backbone of a polypeptide chain.
 * Peptide bond (C1-N): formed from dehydration reaction of COO- and NH3+. Stabilized by partial double bond character from proximal carboxyl group. This also makes it a rigid bond that cannot rotate.
 * Bond from alpha carbon to carbon of peptide bond/carbonyl (C2-C1). Free rotation.
 * Bond from amide nitrogen to alpha carbon (N-C3). Free rotation.
 * Describe the basis for how protein structure determines function and how genetic mutation can affect protein function.
 * Two important models relate protein structure to function: lock and key and induced fit.
 * Lock and Key refers to the idea that the protein has a specific structure that will fit only very specific substrates
 * Induced Fit refers to the idea that when a protein and substrate come together there is a conformational change
 * More generally amino acid sequence determines the structure of the protein. Proteins with similar structures have similar functions.
 * Mutations in even a single amino acid codon can lead to proteins with a different structure, and thus will not perform the same function as well. However, 20-30% of proteins are polymorphic with differences in non-essential amino acids and thus structures that are similar enough to perform the same function.
 * Depending on the specific assortment and order of side chain from the peptide backbone, the pattern of hydrogen- and sulfur-bonds in a protein will be very different. Collagen is a good example, as without the proline residues available for hydroxylation, the strong H-bonds holding it together couldn't happen. Keratin is another example-- without being cysteine-rich, the multiple disulfide bonds that give it its tensile strength would be absent.
 * Re mutations: if the ratio of glycine residues is messed with in collagen (thus deforming the tight helix structure), it results in a fatal condition in infants.
 * Understand that proteases and the specific breaking of peptide bonds can have important functions.
 * Proteases break down peptide bonds through hydrolysis.
 * These can be general proteases such as trypsin, chymotripsin and pepsin. These proteases help in breaking down protein in food during digestion.
 * There are more specific proteases that often help to activate precursor proteins to their active form. In the [|clotting cascade], blood clotting factors are proteases that upon trauma lead to a cascade of activated factors resulting in clots. Angiotensinogen is cleaved by Renin to form Angiotensin I which is cleaved by ACE to Angiotensin II which is the active hormone regulating blood pressure.
 * Describe hydrogen bonds and their role in secondary structure formation.
 * Weak, but numerous, bonds between a hydrogen atom and an electronegative atom such as oxygen. The H is the "donor" of electron density; the electronegative atom is the "acceptor" of electron density.
 * "H bonds between the protein backbone amide nitrogen and carbonyl oxygen are the driving force to form secondary structures."
 * on [|alpha-helices], the H-bonds between every n and n+4 amino acid within the same polypeptide chain, every 8th amino acid is right on top of the 8th before it. These bonds force the chain into a right-handed helix. All side chains point out of the helix.
 * For [|beta-sheets] the hydrogen bonds are between two polypeptide chains (or one chain with a turn). The chains can be parallel or anti-parallel.
 * Describe two major types of protein secondary structures.
 * alpha-helix: ~30% of all protein structures (also see above)
 * All biological alpha-helices are right-handed screws
 * All side chains point towards the outside of helix
 * Every five residues there's a H-bond between the carbonyl O and amine H.
 * Every 8th amino acid is aligned with the one 8 before
 * Hemoglobin is a good example. [|Thalassemia] is a genetic disease where mutations destabilize hemoglobin by disrupting alpha helices.
 * beta-sheet: ~30% of all protein structures
 * Polypeptide chains are held together by H-bonds
 * Strands can be parallel or anti-parallel, anti-parallel is more common.
 * Immunoglobulin or antibody is an example.
 * Notice that the primary structure of the protein determines its secondary structure-- different side groups influence the formation of different patterns of H-bonding.
 * There's also turns and loops to compact proteins (mainly glycine, because it's small and flexible, and proline, because it's slightly kinked).
 * Notice there's a triple helix structure in collagen: different conformation from either alpha-helices or beta-sheets. Has a lot of glycine and proline, like turns or loops, but the proline residues are often hydroxylated (via vitamin C), resulting in massive H-bonding between strands to form the characteristic triple helix.
 * Every three residues there's a glycine residue (the smallest AA, in order to compact the helix).

** Lecture III **

 * Understand what are tertiary and quaternary structures
 * Tertiary structure is the spatial arrangement of the polypeptide chain. The two major classifications of tertiary structure is [|globular] (most of the proteins in the body, lipid or water soluble, diverse functions) versus [|fibrous] (long and composed of alpha helices OR beta sheets, insoluble and have structural or protective role). Separate domains having independent secondary structure can come together to form the tertiary structure.
 * Features which control the tertiary structure of proteins:
 * N-terminus of protein is synthesized first, meaning that the AA's at that end have an opportunity to fold first (affects overall folding and refolding after denaturation).
 * Interactions between adjacent AA's in secondary structures
 * Hydrophobic AA's tend to cluster in the cores of proteins (vs. hydrophilic AA's).
 * Various proteins assist with "chaperone" activity in folding. (about 30% of all eukaryotic proteins are folded by chaperones.)
 * Quaternary structure is how multiple polypeptide chains come together to form a single functional protein. Hemoglobin is made up of four subunits.
 * Understand the role of loops in protein structure and function
 * Enable polypeptide chain to form particular structures instead of staying as extended linear chains
 * Can interact with other proteins such as with variable loops of in [|immunoglobulin] molecule
 * Loops and turns are mostly Glycine and Proline amino acids
 * Understand how to use Kd to represent binding strength
 * Kd is the dissociation constant
 * Kd= [ligand] when 50% of ligands are bound
 * Understand how binding specificity can be achieved
 * Lock and Key complementary model: the ligand and protein have high complimentarity where the ligand fits very nicely onto a site of the protein. There is complementarity of size, shape, charge, hydrophobicity
 * Induced fit model: There is not preformed complementarity (as in lock and key model) but instead both can undergo conformational changes upon binding of the ligand. For enzymes the ligand binds into its transformational state reducing activating energy and speeding up the reaction.
 * Understand how heme enables [|myoglobin] to bind oxygen
 * Free or exposed Fe would be oxidized irreversibly, and the protein itself can’t bind O2. A heme group isolated within the protein works perfectly (without producing free radicals).
 * Myoglobin is the main oxygen storage protein in mammals
 * Understand the molecular basis of carbon monoxide poisoning
 * Carbon monoxide has a similar size and shape to O2 so it also can fit to the same binding site
 * CO binds over 200,000 time stronger than O2 (though the structure of myoglobin and hemoglobin reduce that to ~200:1) so it will out compete O2 every time, blocking the functions of myoglobin, hemoglobin and mitochondrial cytochromes involved in oxidative phosphorylation
 * Understand why [|hemoglobin] is a good oxygen transporter.
 * Myoglobin binds O2 too strongly, so it would not be released in the tissues
 * Hemoglobin has four subunits each with heme groups that interact with each other in positive cooperativity (thus the sigmoidal binding curve). Hemoglobin has a Tense, lower binding affinity state and a Relaxed, higher binding affinity state triggered by initial O2 binding. Thus in the lungs with lots of O2 it will easily bind in the Tense state creating the Relaxed state bringing O2 to the tissues, where the pH is lower and O2 does not bind as well causing it to release and go back to the Tense state. The pH difference is called the Bohr effect.
 * Tense: low affinity state (in lungs)
 * Relaxed: high-affinity state (leaving lungs)
 * High pH = high O2 binding (in lungs)
 * Low pH = low O2 binding (in tissues)

** Lecture IV **

 * Understand the factors that cause protein denaturation.
 * Heat- think of cooking egg whites
 * pH- yeah acids and bases are never good
 * Chemicals- organic solvents, urea, detergent
 * Understand the most fundamental conclusion drawn from the [|Ribonuclease refolding experiment] - the primary sequence of a protein determines its structure.
 * Used reducing agent to denature Ribonuclease A, then after dialyzing out the urea it slowly refolded and restored almost 100% of activity
 * All the information needed to fold the protein correctly is embedded in the primary amino acid sequence
 * The environment provided by the inside of the cell is not always required in order for proteins to fold correctly
 * The protein does not explore all possible structures while folding, there is instead a pathway it follows. (Levinthal’s paradox)
 * Know the two classes of chaperones and the general function of chaperones.
 * Heat Shock Proteins (Hsp70): induced at elevated temperatures and binds to hydrophobic region of unfolded proteins to prevent aggregation, can also help transport some proteins across membranes in unfolded states, works with other heat shock proteins
 * Chaperonin: consists of a cap and two 7-subunit rings. The hydrophobic region of the unfolded protein binds to the hydrophobic region of the chaperonin then with some ATP and a conformational change of the chaperonin the protein is folded at least partially so that it can only continue to the final native shape.
 * Ex: GroEL/GroES complex in E. coli.
 * Understand why sometimes protein disulfide isomerase or protein prolyl isomerases are required for protein folding.
 * Disulfide isomerase: incorrect disulfide bond formation between free cysteines means this enzyme needs to come in, reduce the improper ones and reform them correctly
 * Protein prolyl isomerase: This protein reforms proline from the typical trans formation to the cis formation (6% of proline in mammals) for proper folding and functioning
 * Understand that protein mis-folding is the major cause of prion disease, Alzheimer’s disease, Parkinson’s disease, and amyloidosis.
 * Prion disease - the prion protein (PrP) simply misfolds, which then causes other normal prion proteins to misfold. These are altered from alpha helices to beta sheets causing them to aggregate within amyloid plaques leading to neuron loss and gliosis
 * Alzheimer’s disease : the normal AB-40 folds correctly, however the slightly different AB-42 misfolds and aggregates into amyloid plaques. This leads to B-amyloid plaque and tau tangles.
 * Parkinson’s: Beta-synuclein misfolds into Lewy Bodies.
 * Amyloidosis: Generalized protein misfolding in the rest of the body leading to a variety of disease (from Type II diabetes to Cardiac amyloidosis).
 * Understand the secondary structure changes and the infectious agent in prion disease.
 * The normal prion protein is misfolded into an infectious form. It then converts other normal prion proteins into the bad form. These are altered from alpha helices to beta sheets causing them to aggregate within amyloid plaques leading to neuron loss and gliosis.
 * Describe the major approaches for purifying a protein.
 * According to size by **gel filtration chromatography**:
 * Have proteins run through a filtration column; large proteins flows through quickly, small proteins more slowly.
 * According to charge by **ion exchange chromatography**:
 * Have proteins run through a cation exchange column filled with negatively charged beads; positively charged proteins stick and can be eluted off with salts (more slowly), negatively charged proteins flow directly through.
 * According to ligand binding properties by **affinity chromatography**:
 * Attach a given ligand to beads of column (eg glucose)-- run protein solution through column and only the ligand-binding proteins should stick. Then elute the proteins off the beads into a separate container
 * Ligands: enzyme substrates, DNA, metal ions, carbohydrates, peptides, etc.
 * Extra info for proteins:
 * **Electrophoresis**: (SDS-PAGE) determine size of protein.
 * Apply a detergent to denature proteins and coat them in an even negative charge.
 * Use a polymerized acrylamide gel to separate based on size. Electric field causes denatured, negatively charged proteins to run down gel at a rate dependent on size.
 * Also need to run a "marker" of known molecular weights to give a "size ladder" to determine the approximate size of the protein under investigation.
 * **Mass spectrometry**: determine sequence of (unknown) protein by molecular mass.
 * **Edman Degradation**: Label and remove the N-terminal amino acids one at a time and identify.
 * **Western Blots**: use immunology to identify proteins on an acrylamide gel.
 * Transfer proteins onto a membrane, react with primary antibody for a particular protein, wash off anything unbound, react with fluorescent secondary antibody to detect.
 * One use is to identify HIV infection: use patient's serum as primary antibody (checking for HIV antibodies in serum) against HIV proteins.

** LO’s not specific to C/O 2013 **

 * Know the reasons why drugs are more often used to inhibit the interactions of small molecules with proteins rather than protein interactions with other proteins
 * Protein-protein interactions tend to occur over large areas of the protein(s). Thus it's tough to inhibit efficiently-- but protein-small molecule interaction occurs with a very punctiliar character and can be targeted efficiently.
 * Also, the primary AA sequence of small ligand-binding domains can be studied and drugs designed to interact specifically with that area and no others (the larger the surface area of affect, the larger the chance of unintended interactions).
 * Usually antibodies are used to deal with protein-protein interactions.
 * Know the five different ways in which proteins are modified after synthesis
 * Disulfide bonds can be formed and modified with protein disulfide isomerases.
 * Prolyl isomerase catalyze rotation of proline residues from //cis// to //trans// or vice versa.
 * Restricted cleaving by proteases can "activate" various proteins (cleave off the portion that keeps it inactive).
 * Glycosylation of secreted and cell surface proteins
 * N-linked: added to asparagine (-X-**N** -X- **S/T** -)
 * O-linked: added to serine or threonine
 * Various other non-genetically coded factors: metal ions, vitamins, etc.

=**Mitosis and Cell Cycle**=

** Lecture I **

 * Understand how cells regulate their size by coordinating growth with division at the restriction point ("R") in G1 phase.
 * In somatic cells, there's a "R point," or restriction point, at which the cell has to make a decision - based on whether or not various hormones or growth factors are there, as well as whether the cell is large enough - whether or not to undergo replication and mitosis. (checkpoint before leaving G1 and entering S)
 * "R" point = first (thus most highly regulated) step in cell cycle pathway.
 * In embryonic cells the R point is bypassed and continues to divide despite getting smaller every time. At all other times the R point prevents reducing the surface to volume ratio, "divide itself out of existence.”
 * Know that the main goal of the somatic cell cycle is to ensure exact duplication of the genome in S phase followed by exact of division of the genome in M phase to produce identical daughter cells.
 * Don’t want extra chromosomes (reinitiation), etc., want them divvied up correctly
 * Know how cells prevent re-replication of their genomes by keeping the assembly and activation of replication complexes in separate cell cycle phases.
 * **M**- high CDK prevents building Pre-Replication Complex
 * **G1**- low CDK allows building of Pre-RC
 * **S**- high CDK activates replication and prevents any more Pre-RC building
 * During G1:
 * Orc proteins [Origin replication complexes] initiate replication by binding to the DNA origin and binding to it; other proteins (cdt1 and cdc6, helicase) complex with Orcs.
 * In short: pre-RCs assembled (but not activated) during G1, and activated (but not assembled) during S, driven respectively by the low and high concentrations of CDK in the cell.
 * Know that genomic instability either by chromosome re-replication in S phase or mis-segregation during mitosis produces human disease such as cancer and birth defects ([|Trisomy 21] ).
 * You never want excess or shortage of genetic material as it will be translated…

** Lecture II **

 * Compare the cell cycle of somatic cells (mitosis) with that of germ line cells (meiosis), which produces haploid gametes.
 * End point of mitosis: 2 identical diploid cells from 1 diploid cell.
 * End point of meiosis: 4 different haploid cells from 1 diploid cell.
 * Start: chromosome A and chromosome a.
 * 1st: replication of chromosomes (AA, aa)
 * 2nd: homologous recombination of chromosomes to provide genetic variation
 * 3rd: separate homologues (diploid to diploid, or meiosis I) (one cell: AA; another cell: aa).
 * 4th: separate chromosomes of daughter cells (diploid to haploid, or meiosis II) (two cells: A, two cells: a).
 * Know that differentiated, post-mitotic cells such as neurons are stuck at the "R" point in that they continue to grow without cycling.
 * These cells need to grow and serve specific long-term functions (all those stable synapses)
 * Know how alterations in many cell cycle regulators that are found in cancer cells are being used for patient diagnosis and prognosis.
 * Diagnosis: sequence genes as with LFS (ATM mutation), BRCA, etc. for specific, common mutations
 * In general:
 * CDKs [cyclin-dependant kinases] : 6 enzymes central to regulating the cell cycle that phosphorylate Ser, Thr, Tyr and are regulated by cyclin
 * Active CDKs are produced by growth factor hormones (and cancer factor proteins) and result in cell replication and duplication.
 * Particular CDKs tend to be found at particular levels in various types of cells. So if an elevated level of a given CDK is found in a tumor, that's an indication of active proliferation.
 * Low levels of RB, or highly phosphorylated (inactivated) RB will indicate abnormally high replication rates (as it inhibits entrance into S)
 * There's also a family of proteins called CDIs [CDK Inhibitors] that turn off CDKs (thus repressing the cell cycle]. Mutations in these proteins often turn off the ability to turn off a given CDK, thus result in uncontrolled proliferation.
 * Most frequently seen in cancer cells: mutation in Ink4 (inhibitor of CDK4) CDI family that lets CDK4 push past the "R" point regardless of other growth factors.
 * So can look for mutations in CDI genes as a cancer marker as well.
 * **mitogen** : signal protein that leads to activation of CDK4. Notice that this leads to a positive feedback cycle which leads to the irreversible "commitment" to completing the cell cycle.
 * Understand the importance and the mechanism of cell cycle checkpoints in maintaining genomic stability.
 * [|Cell cycle diagram] from Wikipedia
 * Checkpoint at G1: Is the cell large enough? (R point/Rb pathway)
 * Checkpoint at S: Should DNA be replicated? Any mutations? (S point/p21 on cyclinE)
 * Checkpoint at G2: Correct copies of new/old DNA?
 * Checkpoint at M: Did the spindles form and act as they should?
 * Mitogen produces CyclinD which inhibits Rb popping off E2F creating cyclinE which initiates S phase. p16 (Ink4) inhibits CDK4,6 preventing passage to S phase (R point).p21(Cip/Kip) inhibits CDK2 preventing activation of synthesis (S point).
 * Failing any of these checkpoints results in a cessation of the cell cycle (cell stays in phase it's in when the checkpoint's failed) until the damage is repaired or the checkpoint is passed. Notice that you can repair DNA in any phase.
 * If the damage can't be repaired, have apoptotic signaling.
 * Mechanism: Sensor proteins on the DNA communicate with transducers which communicate with effectors that regulate the cell cycle via checkpoints.
 * Summary: sensors (such as Rad 17) -> ATR ->Chk1,2 activating repair enzymes and BRCA1/p53 (respectively) OR ->ATM ->p53 -> p21 leading to arrest, apoptosis, DNA repair
 * Notice that most cancer is not inherited unless the mutation is present in germ cells. (See 2-hit hypothesis under Li-Fraumeni Syndrome below)

=**Enzyme Kinetics**=
 * Be able to describe what an enzyme is, the characteristics of enzymes, and thermodynamically how they increase the rate of a reaction. Be sure to understand the terms: catalyst, activation energy, free energy of the reaction.
 * Enzyme: molecule that increases the rate of a chemical reaction without itself being irreversibly changed in the process
 * Have specific structures and active sites where the catalysis happens
 * Some use specific cofactors or coenzymes that help in the catalysis
 * Classified and named by the reaction that they catalyze
 * Catalyst: something that increases the rate of a reaction (such as an enzyme)
 * Activation energy: the hump of increased energy that must be overcome for products to become reactants, dictates the **rate** of the reaction.
 * Reaction free energy: free energy differential between reactants and products (products – reactants) dictates the **spontaneity** of the reaction
 * Be able to describe how enzymes work, using the terms: binding of the transition state, induced fit, covalent chemistry, metal ion chemistry, general acid-base chemistry.
 * What you're essentially talking about is the following equation: E + S -> ES -> EP -> E + P, where E = enzyme, S = substrate, and P = product.
 * Current theory: Enzyme binds and stabilizes the substrate in its transitional state, where induced fit can change the conformation of the substrate and/or enzyme putting even more pressure on the substrate towards becoming the product. This decreases the amount of energy (activation energy) needed to form the product.
 * Active site chemistry can rearrange covalent bonds
 * Metal ion chemistry refers to the idea that some enzymes utilize cofactors that attract the transition state and help to form the substrate product via redox reactions (almost 1/3 of enzymes)
 * General acid-base catalysis: side chains of enzymes donate or accept protons to stabilize transition states
 * Covalent catalysis: transient covalent bond between enzyme and substrate
 * Define the terms cofactor and coenzyme.
 * **cofactor** : Metal ions required for enzymatic activity.
 * **coenzyme** : An organic ligand that binds to the enzyme and allows it to act on the substrate.
 * Some terms associated with this:
 * A ligand that is tightly or covalently linked to the enzyme is a **prosthetic group**.
 * The complex of a cofactor or coenzyme with an enzyme is a **holoenzyme**
 * The enzyme dissociated from its cofactors or coenzymes is an **apoenzyme**.
 * Understand the significance of the terms Km and Kcat (know what it means if one enzyme has a lower or high Km than another, etc.). Be able to estimate the value of Km from a graph of reaction velocity versus [S].
 * **Km**:
 * Km is the substrate concentration at which the 'enzyme velocity' of the reaction is 1/2 the maximum velocity of the enzyme.
 * Generally, enzymes in biological systems tend to operate around ½ Vmax, as this is the typical substrate concentration in the cell.
 * If an enzyme has a lower Km than another, then it needs less substrate to operate at ½ Vmax
 * [|Lineweaver-Burke plot] (Double-reciprocal plot) : a way to empirically figure out Km and Vmax.
 * **Kcat**:
 * kcat : "turnover number", a rate constant for a given enzyme of how many substrate molecules are converted to product in a given unit of time by a molecule of that enzyme //under saturated conditions//.
 * Kcat/Km tells you the overall efficiency of an enzyme
 * Smaller Km = less substrate needed to work at ½ Vmax. Bigger kcat: faster action of enzyme. Thus a large Kcat/Km is indicative of a highly efficient molecule.
 * Describe 4 different types of inhibitors and, in general, how each works (competitive, uncompetitive, mixed, irreversible).
 * **Reversible** inhibitors: Binds to enzyme and decreases enzyme function until released.
 * **Competitive** inhibitor: inhibitor goes into the substrate binding site. Competes for the binding site with the normal substrates. Increases Km.
 * **Uncompetitive** inhibitor: inhibitor binds somewhere other than the binding site. Means it doesn't have to compete with normal substrates for binding. Only binds to the ES complex. Lowers Vmax and changes Km (note that Noncompetitive is //not// the same - it is a type of mixed inhibitor)
 * **Mixed** inhibitor: inhibitor binds outside of active site to either E or ES complex, affects both Km and Vmax.
 * You get different kinds of Lineweaver-Burke plots for these different types of reversible inhibitors. So you can figure out experimentally what kind of inhibitor you're looking at by using these plots.
 * 4. **Irreversible** inhibitors: combine with or destroy a functional group of the enzyme. This is a permanent change.
 * Ex.: Penicillin irreversibly inhibits enzyme responsible for polymerizing peptidoglycans, weakening bacterial cell walls. Goes into active site and forms a covalent, irreversible complex with enzyme.
 * Describe 4 types of enzyme regulation mechanisms and, in general, how each functions (allosteric, covalent modification, binding of another protein, proteolytic cleavage).
 * Basic principles: generally the first enzyme in a long regulatory pathway is the one most highly regulated.
 * 1. **Allosteric regulation**: Some positive or negative modulating molecule binds to the enzyme and causes a conformational change to allow or disallow the action of the enzyme on the substrate.
 * Sometimes this is involved in biofeedback loops where the end product of the pathway is the allosteric inhibitor/activator of the first enzyme in the pathway (neg, pos feedback).
 * 2. **Covalent modification** of the enzyme:
 * Many enzymes can be phosphorylated to dramatically affect functionality.
 * The enzymes that modify enzymes to turn them on or off are usually called **kinases**.
 * Also a variety of other modifications: adenylations, methylations, etc.
 * 3. **Regulatory protein binding**: Some enzymes are bound by proteins to activate or inactivate them.
 * 4. **Proteolytic activation**: Some enzymes are inactive until cleaved by a particular enzyme (ie trypsinogen doesn't become active until it gets to the duodenum and is cleaved to trypsin), (angiotensin-renin pathway).

=**Tools of molecular biology**=
 * Write out any three double stranded DNA sequences that are likely to be cut by restriction endonucleases, i.e. palindromic (a known restriction enzyme does not have to exist for your sequences).
 * Palindromic usually means they read the same forwards and backwards (ie "A man, a plan, a canal, Panama"). Here it seems to mean that the same sequence, read 5' to 3' on the two complementary strands (NOT on the same strand) will read the same.
 * Here's our sequence: 5' TACGTA 3'. This implies its complementary sequence: 3' ATGCAT 5'. If you read the 5' to 3' sequence on each, it's the same. Note that TACGTA is not a classical palindrome (it doesn't, by itself, read the same backwards) so don't get confused.
 * Some other sequence examples:
 * TTTAAA
 * TCCGGA
 * CTATAG
 * Explain the principle of [|electrophoretic separation] of DNA to a premed student.
 * You want a method to separate DNA strands by size. You do it with a polarized electrical field in a gel that will exert a uniform electromotive force on all the DNA in that gel to move down its gradient.
 * The gel is thick but porous, meaning that smaller strands of DNA will move through it faster than larger strands. It's made of polymerized acrylamide (or other stuff)
 * Because DNA has a uniform negative charge per unit length (from the phosphate backbone) only size of the strand affects its speed.
 * The DNA samples are placed in wells in the gel, and a current is applied - the DNA will migrate toward the positive pole. A "ladder" mixture containing known sizes of DNA is run alongside the samples for comparison.
 * Remember: //Smaller strands migrate farther down the gel than larger ones//
 * A substance that intercalates into the DNA and fluoresces, such as ethidium bromide, is used to visualize the results.
 * A [|Southern Blot] is performed by denaturing the DNA then blotting the gel (after electrophoresis) onto a membrane that binds DNA. The membrane is then washed with short, labeled "probe" sequences of DNA or RNA, and visualized.
 * This technique is used when examining a large quantity of many types of DNA for particular sequences, e.g. for diagnosis of genetic diseases.
 * Give an example of a disease that can be diagnosed using a restriction fragment length polymorphism (RFLP) and a use of DNA fingerprinting. Describe at least three experimental stages required in each of these procedures.
 * RFLP: Example of looking for the HbS mutation of sickle cell anemia.
 * Sickle Cell anemia results from a mutation that, coincidentally, lies in a restriction site.
 * Digest patients' DNA with diagnostic restriction enzymes (MstII)
 * Electrophorese against a normal genome
 * Southern blot with P32 labeled B-globin gene- if 1.1kb and 0.2kb then normal, sickle cell is both together at 1.3kb
 * DNA fingerprinting: Example: paternity testing
 * PCR with primers that surround variable number tandem repeat (VNTRs) sequences
 * Electrophoresis/detection of altered size of DNA fragment patterns.
 * Compare sample against target(s) for similarity
 * List the names given to the transfer of DNA, RNA and protein respectively from an electrophoresis gel to a membrane. Describe three characteristics of a hybridization probe that you will use to detect a specific DNA sequence on a membrane.
 * Transfer of DNA: **Southern blot** (actually named after somebody)
 * Transfer of RNA: **Northern blot** (to follow Southern)
 * Transfer of proteins: **Western blot** (to follow Northern)
 * Characteristics of probe:
 * Specific length: you need to know what temperature to anneal the probe to the DNA with, and that's dependent on the primer's length.
 * Specific sequence: so it can bind to the DNA you're interested in.
 * Radioactivity (or some other visualization characteristic): so you can detect it later.
 * Quantity: add it in sufficient quantities to outcompete the other strand of DNA in annealing to its target DNA.
 * Recall the classes of enzymes that are used in recombinant technology to:
 * a) copy a DNA sequence into a DNA sequence: DNA polymerases.
 * b) copy an RNA sequence into a DNA sequence: reverse transcriptases.
 * c) join DNA fragments: DNA ligases.
 * Describe the three main stages that are repeated multiple times during PCR amplification. State the approximate temperature of each step, and relate this temperature to the state of the DNA molecules in the PCR reaction.
 * Step 1: add thermal-stable DNA polymerase (usually Taq polymerase) and dNTPs (deoxyribonucleoside triphosphates) and desired primers.
 * Step 2: Heat to 95 degrees C (denaturing DNA).
 * Step 3: Cool to 55 degrees C (allows primers to hybridize).
 * Step 4: Warm to 72 C at which time the polymerase copies the DNA.
 * Describe at least 1 distinct use for PCR amplification in the diagnosis of a genetic condition in your patients.
 * Prepare a primer that hybridizes with a mutant copy of a gene but not the normal copy. Then hybridize and replicate as per normal PCR with your patient's chromosomes. PCR will generate a detectable amplification of genetic material if the mutant gene is present. Thus can use PCR as a diagnostic test for discovering mutant genes that are markers for cystic fibrosis, beta-thalassemia, etc.
 * Compare and contrast the molecular details of the processes of DNA sequencing and PCR amplification in a short paragraph.
 * DNA sequencing: uses a primer for a given start sequence (at the beginning of the region to be sequenced) and four differently labeled dideoxynucleic acids, as well as normal unlabeled dNTPs, in DNA synthesis. When the DNA synthesis incorporates a ddNTP, it stops synthesis with a particular final "color" or label, indicating which base it ended with. You run this a while and allow a lot of different lengths of DNA to be made. The product is then run through a column chromatograph that separates based on size. A detector at the bottom of the gel detects the different colors of labeled ddNTPs as they flow past. (In the "manual" version, labeled ddNTPs would be run in a reaction in four separate tubes, on for each kind of ddNucleotide, then run in four adjacent rows with gel electrophoresis)
 * PCR amplification: uses a primer for a given start sequence and DNA polymerase to make more copies of a desired DNA sequence. Use varying temperatures to melt DNA, hybridized probe, and have polymerization reaction happen.
 * The point of PCR is to amplify (make lots of copies of) a given DNA segment. The point of sequencing is to determine the sequence of an unknown DNA fragment.
 * Similarities: Both use primer sequences to initiate replication of genes.
 * Differences: PCR is used on double-stranded DNA, sequencing on a single-stranded DNA fragment. Sequencing used ddNTPs, PCR doesn't.
 * Be aware of the different types of cloning vectors and their general features.
 * Plasmids: Vectors for amplifying DNA sequences in bacteria, max insertion of 20kb into E. coli, simple but inefficient.
 * Bacteriophage: Used to infect //E. coli// and use its replication machinery to produce the recombinant vector. Insertion up to 25kb, more efficient than plasmid.
 * Cosmids: hybrid of bacteriophage and plasmid: use plasmid replication origin; can take up to 45 kb insert into E. coli.
 * BAC: bacterial artificial chromosome with insert up to 300kb, good for chromosome mapping and sequencing
 * YAC: yeast artificial chromosome with insert up to 2mb, chromosome mapping and sequencing
 * Retroviral vectors: can carry very large inserts; introduce DNA into mammalian cells, delivers gene therapy.

=**Clinical vignettes**=

**Alzheimer's**

 * Review Pathophysiology underlying [|Alzheimer’s disease] (AD)
 * A β -42 is the incorrectly cleaved protein that incorrectly folds and becomes insoluble and leads to the amyloid plaques
 * Tau protein also forms fibrillary tangles
 * The tau and A β plaques leads to inflammation and neuronal loss
 * Sources:
 * Apolipoprotein E4 increases amyloid deposition
 * ApoE2 decreases amyloid
 * Mutations can preferentially lead to A β -42 cleaving by blocking alpha or gamma secratase sites, or easier to cleave the beta site
 * Presinilin 1 mutation on chromosome 14 increases A β -42 production via gamma secretase
 * Presinilin 2 mutation on chromosome 1 also increases A β -42
 * Extra chromosome 21 increases all APP products
 * Understand the central role of post-translational amyloid precursor protein processing in the pathogenesis of AD
 * Consider interventions that might help delay onset or prevent development of AD
 * Inhibit all APP products
 * Antibodies that attack A β -42

**Prion disease**

 * Explain the relationship between prion protein (PrP) and infectious prions, at the level of protein structure
 * PrP is mostly alpha helices, infectious prions are full of beta-sheets. These beta-sheets are more hydrophobic and clump into lesions.
 * Describe how prion disease can be sporadic, inherited or infectiously acquired
 * Sporadic- random misfolding that infects the rest of the prions
 * Inherited- familial strains that lead to early onset [|Creutzfeldt-Jakob Diseas] e, fatal insomnia
 * Infectious- can be from diet (vCJD or Kuru) or iatrogenetic (from medical treatment- pituitary hormones and dura mater)
 * Describe the relationship between bovine spongiform encephalopathy and the disease of humans known as “variant Creutzfeldt-Jakob disease”
 * BSE is mad cow disease and vCJD is a strain of prion disease that originated in cows and through dietary ingestion led to vCJD in human (much lower incidence and 5-10 year lag compared to CJD).
 * Explain what is meant by the concept of “prion strains”
 * A prion strain has distinct characteristics such as incubation time in defined host, clinical signs, distribution of protease resistant PrP in the brains of affected animals. These features are stable on serial propagation. Note that all strains are have the same sequence (as PrP)
 * Ex: Hyper and Drowsy strains of PrPsc as tested in hamsters. The varying characteristics are incubation time (70 v 148 days), symptoms (hyper v drowsy), distribution of histological lesions, distribution of prions in brain

**Li-Fraumeni Syndrome**

 * Describe criteria for classifying hereditary cancer syndrome as LFS
 * [**proband** : patient reported, diagnosed, or treated]
 * sarcoma in proband before 45 years of age
 * 1st degree relative with any cancer under 45 years of age
 * 1st or 2nd degree relative with any cancer under 45 years of age or a sarcoma at any age
 * Describe [|Knudson "two-hit" hypothesis]
 * One p53 allele is mutated already at birth. The other needs to get knocked out at some point along the way to cause the cancer.
 * Notice that mutation in another gene, instead of the p53 gene, may be enough to cause cancer as well.
 * Describe the function of p53 in response to UV exposure.
 * p53 protects body from the damaging effects of radiation exposure; thus a mutation in p53 can lead to susceptibility to radiation damage (which is also why p53 mutation-caused cancers aren't treated with cancer radiation therapy).
 * p53 arrests the cell cycle to allow DNA to repair itself.
 * p53 also causes apoptosis if excess DNA damage.
 * General Characteristics of LFS:
 * rare autosomally inherited cancer susceptibility
 * large age range of presentation
 * must have more than one family member with cancer to diagnose
 * over 250 mutations in p53 gene
 * often test "hot spots" in p53 gene (in DNA binding domains) rather than all 2300 bps