BCHM465 Final
What is the evidence against the RNA world hypothesis? (11) *be able to list at least 5
1. Modern cells use RNA/DNA protein machines 2. Purine/pyrmidine NTP enzymatic synthesis involves 11/6 steps, 11/6 protein enzymes, several protein kinases, and cofactors 3. Nucleic acid building blocks require wet and dry cycles, problematic for unsupervised reactions 4. Unsupervised (w/o proteins) prebiotic synthesis of RNA components prone to producing "tar" 5. Reactivity of 5'-phosphate and 2'-OH makes prebiotic synthesis of 3'-5' linkage problematic (5'-5' or 2'-5' linkage more likely) 6. Ribose has short half-life (hours at 100 degrees C), precludes its use as prebiotic molecule 7. Hot early earth would lead to decomposition of all 4 nucleobases 8. A and G minimim decomposition rate as a function of pH is limited -for A, optima ~4-10 -for G, optima ~5 9. Many sites of thermodynamic instability in RNA are hydrolyzed in water 10. Mixed phosphoanhydride bond of aminoacyl-tRNA is unstable at neutral and alkaline pH 11. Phosphodiester bond stable at acidic pH, ~4 at 90 degrees C
Describe the order of events where the snRNPs bind mRNA. (7 steps)
1. U1 binds at 5'SS 2. U2 binds at BP (A) 3. U6/U5/U4 tri-snRNP displaces U1 from 5'SS 4. U5 binds 5'SS and 3'SS 5. U6 dissociates from U4 6. U6 forms intramolecular stem loop and base pairs with U2 7. U4 is displaced
What is the evidence for the RNA world hypothesis? (6)
1. We can make all 4 bases in lab under conditions that appear pre-biotic (4500 K and 650 K, atmospheric gases, NH3, CO) -but there are concerns for stability of bases and rNTPs 2. Discovery of various functional RNAs involved in gene regulation 3. RNA fragments attached to various cofactors suggest RNA priority (e.g., ATP, SAM, FAD, NAD, Coenzyme A, Biotin) 4. X-ray structure suggests ribosome is a ribozyme, proteins not needed 5. Ribosome devoid of proteins can still form a peptide bond in a model system of puromycin and Fmet-tRNA oligo 6. Discovery of ribozymes: self-cleaving, self-splicing, riboswitches -group I intron is RNA enzyme -RNAseP RNA component is an RNA enzyme
What are the two mechanisms of the riboswitch that binds TPP?
1. When bound to TPP, the riboswitch assumes a conformation that "hides" the Shine-Dalgarno sequence by base-pairing with it --> ribosome can't access it and start translation 2. Splicing regulation -riboswitch is part of an intron -there are two GU's (two 5'SS) -under low TPP, the first 5'SS is used --> short mRNA -under high TPP, the second 5'SS is used --> long mRNA
Explain how the ribosome recognizes the codon-anticodon helix: what surface is recognized?
3' end of 16S rRNA forms interaction w/codon and anticodon - near cognate loses key interactions w/16S rRNA elements -near cognate less tighly packed, loses key interactions Whenc orrect tRNA binds, shoulder lifts up and closes
What is the structural basis of high fidelity of the ribosome (generally)?
3' end of 16S rRNA forms interactions w/codon and anticodon -near cognate is less tightly packed, loses key interactions with 16S rRNA elements in the minor groove Ribosome sense shape at the decoding center -binding at the feet dictates orientation of the arm (correct - arm fits into ribosome)
Be able to draw/recognize the following: 1. Structure of cylic or dicyclic nucleotides 2. Mechanisms for spliceosome, group I and group II self-splicing introns 3. Anti vs. syn conformation of nucleotides 4. The WC base pairs 5. Amounts of maternal and zygotically encoded DNAP throughout development 6. Polymerization reaction during DNA replication and transcription 7. Base-catalyzed RNA hydrolysis 8. Peptidyl transferase reaction during translation (and the peptidyl-tRNA hydrolysis reaction) 9. Dihedral angles along DNA/RNA backbone
:)
How does the ribose pucker prfoundly impact the 3D structure of nucleic acids?
A-DNA is C3' endo, while B-DNA is C2' endo Sugar pucker affects phosphate-phosphate distance in the backbone For C3' endo, distance is 5.9 angstroms; for C2' endo, distance is 7.0 angstroms Affects how close together the bases are
What are the distances between base pairs for A-DNA and B-DNA in angstroms?
A-DNA: 2.4 B-DNA: 3.4
What can you conclude about the requirement of GTP/ATP for transfer of charged tRNA to the ribosome in the Hoagland experiment?
ATP is sufficient for charging the tRNA molecules with the labeled amino acids -extent of labeling increased with [ATP] GTP is necessary to transfer the labeled amino acids to the ribosome -in the absence of GTP, the amino acid-labeled RNA decreased, but the appearance of amino acid in protein did NOT increase
At what points in the translation cycle is GTP/ATP needed?
ATP needed during tRNA charging 2 GTP molecules needed during elongation 1. Aminoacyl-tRNA binds EF-Tu-GTP --> brings charged tRNA to A site (GTP hydrolysis is needed for proofreading and accommodation 2. Translocation requires another GTP
What is the difference between specificity and affinity? What is Kd?
Affinity is how tight the binding is; specificity has to do w/recognizing the correct substrates Kd is a good marker for affinity
The following experiments by Avery et al idnicate the necessity of DNA as genetic material for all organisms: T or F a. only RNAse treated extract was incompetent to transform avirulent to virulent pneumococus bacterium b. only DNAse treated extract was incompetent to transform avirulent to virulent pneumococus bacterium c. only protease treated extract was incompetent to transform avirulent to virulent pneumococus bacterium d. only protease treated extract was competent to transform avirulent to virulent pneumococus bacterium e. only DNAse treated extract was competent to transform avirulent to virulent pneumococus bacterium
All are false except b
The sequence hypothesis of molecular biology is: T or F a. a negative statement that says info transfer from proteins to nucleic acid exists b. a positive statement that says information transfer from nucleic acid to proteins exists c. a negative statement that says information transfer from proteins to nucleic acid does not exist d. a positive statement that says information transfer from proteins to nucleic acids exists
All are false except b
True or False: Three key components of monomer units for building nucleic acids are a. ribose, pyridine, phosphate b. deoxyribose, purine base, phosphate c. ribose, adamantine, phosphite d. deoxyribose, purine base, pyrimidine base e. none of the above
All are false except b
Describe the overall process of rNTPs coming into the E and A site of RNAP. What happens when the correct rNTP enters?
All nucleotides bind transiently to the E-site in open complex Once the correct nucleotide enters, isomerization to the A-site and positioned for catalysis -positioned by Mg2+ ions Bridging helix "shuts the door" and traps the nucleotide in the A-site
What do the following subunits of DNAP do? Alpha, epsilon, theta, and beta
Alpha = polymerization activity Epsilon subunit = 3'-->5' proofreading exonuclease Theta = stabilizes epsiolon Beta clamp = DNA clamp required for optimal processivity; helps polymerase not fall off the DNA
Describe the process of charging tRNA molecules. What enzyme carries out these reactions? Why is ATP required?
Aminoacyl-AMP intermediate has energy stored in the bonds that drives subsequent reactions forward Aminoacyl-tRNA synthetases carry out the reactions Enzyme binds AA and joins it to AMP by cleaving two phosphate groups from ATP -carboxylate of the AA attacks the alpha phosphate of ATP -forms an aminoacyl-AMP intermediate Correct tRNA binds the enzyme, AMP is released Class I enzymes attach the AA at the 2' position first, then transfer it to the 3' position; Class II enzymes attach the AA to the 3' position to begin with
RNAP: Discuss the role of the highly conserved aspartic acids and magnesium ions, the role of the exonuclease domain, and ability to discriminate correct from incorrect rNTPs and dNTPs using recognition of minor groove structural elements, [MM1] specific 2'-OH groups, etc.
Asp residues coordinate Mg2+ ions -one Mg2+ binds the 3'-OH of the growing chain and the alpha phosphate of the incoming NTP -other Mg2+ helps coordinate alpha, beta, and gamma phosphates of incoming NTP for inline attack Exonuclease activity of RNAP is not a separate domain like in DNAP Trigger loop helps recognize rNTP's via interactions with minor groove elements = ribose 2' OH group!
How does what existed in the cell, before the zygote is able to make its own DNA/RNA/Protein, affect the developmental program of the zygote?
At the moment, we don't know if early maternal DNA polymerase has any subtle effects This is an ongoing area of research Do we need to think of the cell code, not just the DNA code
Using tabulated values of changes in enthalpy and entropy, explain whether base pairing is enough or not enough to math expected difference in free energy for fidelity of DNA replication
Base pairing is NOT enough
How are group I and group II intron ribozymes similar to the spliceosome?
Both use the 3'-OH of the 5'-exon to serve as the nulceophile that attacks the 3'SS and joins the 5'- and 3'-exons together Also, for group II - the entire reaction mechanism is the same (the 2'-OH of the branch point A is the nucleophile that attacks the 5'SS) Also, all require Mg2+
Describe the 2-step mechanism of splicing catalyzed by the spliceosome.
Branch point (A) is highly conserved - normally bulges out of the intron 2'-OH of A attacks the 5'-SS junction --> creates 2'-5' linkage 5'-exon gets kicked off but doesn't leave the spliceosome 3'-OH of exon 1 (usually the last nucleotide is a G) gets deprotonated - attacks at the AG junction Forms phosphodiester bond between 5' and 3' exons
What is the chi torsion angle? How does it affect overall structure?
Chi torsion describes the angle between the sugar and the base Defined by 4 atoms in a plane -for pyrimidines: O4'-C1'-N1-C2 -for purines: O4'-C1'-N9-C4 Syn is when the bulk of the base is over O4' (not favorable) Chi-torsion affects C1'-C1' distances -when both are in the anti-position, C1'-C1' distance is fixed (structural basis for Chargaff's rule)
What are the two mechanisms of action of the riboswich that binds SAM?
Cis transcription termination Trans translation repression
What is the structural basis of high fidelity in regard to codon sampling, codon recognition, GTPase activation, GTP hydrolysis, EF-Tu domain re-arrangement, accommodation?
Codon sampling -complex of AA-tRNA-EF-Tu-GTP brought to A-site codon until a match is detected Codon recognition -if anticodon matches the mRNA codon --> shoulder lifts up and closes GTPase activation -3'-end of the aa-tRNA is distorted (recognition at the feet of the tRNA in the small subunit translated to this 3' distortion at the "head" of the tRNA in the large subunit) -GTP provides the phosphate that anchors/holds everything in place -when GTP is hydrolzed, EF-Tu loses affinity for large and small subunit EF-Tu domain rearrangment -EF-Tu-GDP loses grip on Sarcin-Ricin Loop of the 23S rRNA and also the 3'-end of the aa-tRNA Accommodation -strained aa-tRNA springs into peptidyl transferase center (cognate has better interactions) or dissociates from the ribosome (near-cognate has weaker interactions - rejection)
Explain based on active site geometry, conserved amino acids, and the role of Mg2+ how the DNAP active site aligns the correct dNTPs for catalysis?
Conserved Asp residues help align Mg2+ ions Mg2+ coordinate to the 3'-OH group of nucleotide 1 and the phosphates of nucleotide 2 to align them for inline attack on the alpha phosphate by the 3'-OH Conserved tyrosines act as steric gate to exclude dNTPs (steric and electrostatic)
What evidence suggest the info contained in the genome code of an organism, without the cellular environment, is insufficient to make that organism?
DNA + 0 = 0 What this means is that if we just have DNA in the zygote or any cell, nothing will happen - no life will form We still need proteins/cellular machinery to actually have anything happen We already know from Venter's experiments that DNA alone is insufficient The zygote has the DNA, but it lacks the protein machinery to translate the DNA into RNA and protein
Based on the 3D structure of the DNAP reaction, determine where DNAP binding occurs (which face of DNA). Given what you know about B-DNA widths for major and minor grooves, explain how DNAP is able to bind into a particular groove.
DNAP binds into the minor groove (which is narrow and shallow) DNAP binding --> widening of the minor groove DNAP distorts B-DNA to widen the groove, allowing for optimal recognition
What characteristics of DNA-dependent DNA synthesis is NOT the same for DNA-dependent RNA synthesis? (3)
DNAP has a separate exonuclease domain, RNAP does not have a separate domain - instead, has a "tunable" active center Also...RNAPs discriminate rNTPs by binding the 2'-OH group; DNAPs discriminate dNTPs from rNTPs using steric clash and repulsion of the 2'-OH group of the ribose through conserved tyrosine residue DNA synthesis requires a 5' RNA primer, RNA synthesis does not
What is the function of the following proteins? DnaA DnaB DnaG SSB DNA gyrase/topoisomerase
DnaA = recognizes ori sequence, opens duplex at specific sites in the origin DnaB/helicase = unwinds DNA -hexamers of DnaB bind each strand DnaG/primase = synthesizes RNA primer SSB = single-stranded DNA-binding protein that binds ssDNA and prevents it from re-annealing DNA gyrase/topoisomerase = relives torsional strand generated by DNA unwinding by creating small nicks in backbone
What will be the effect of EDTA on polymerase reactions?
EDTA is a chelating agent - would bind the Mg2+ ions The polymerases would not be able to function effectively without these ions - they are metalloenzymes!
What is the accurate order of events in translation elongation?
EF-Tu-GTP escrots various aminoacyl-tRNAs into A site GTP hydrolysis allows for proofreading and accommodation -releases aminoacyl end of tRNA --> correct codon-anticodon interaction rotates tRNA into correct position for peptidyl transferase activity (incorrect aminoacyl-tRNA rejected) Transpeptidation -aminoacyl-tRNA in A site performs SN2 type reaction; amino group attacks C terminus of growing AA chain --> kicks off the tRNA -growing peptide chain is in the A site, P site just has tRNA-OH -translocation of tRNA-OH to E site, growing AA chain moves to P site
What is the RNA world hypothesis?
Earlier life forms may have used RNA as their information molecule RNA appears to possess self-replication and catalysis - RNA could have allowed primitive systems to function w/o DNA or protein
Below is the information about ribose: -decomposes between pH 4-8 from 40-120 degrees celsius -short half life of 73 minutes at pH 7 and 100 degrees C -half life of 44 years at pH 7 and 0 degrees celsius Explain how ribose could or could not be component of the first genetic material.
Earth would have to be ice cold An information molecule must be stable so the genetic inforamtion can be transmitted faithfully to progeny
What atom substitutions are different between dF and dT, for dZ and dA? What happens if the template base is A? What happens if the tempalte base is Z? When can dATP be incorporated (what base(s) can be in the template)? When can dZTP be incorporated (what base(s) can be in the template)? Explain how this results in light of replication mechanism. How would exonuclease domain affect these experiments?
F is an analog of T, except it has fluorines instead of the carbonyl groups Z is an anlog of A except it has a methyl group instead of the amino group If the template base is A --> T mostly incorporated (sometimes F) If template base is Z --> F mostly incorporated, sometimes T dATP can be incorporated when T or F is in the template, but dZTP is only incorporated if F is in the template (will not get incorporated across T) Conclusion: shape is very important, not just hydrogen bonding -shape of enzyme active site contributes to fidelity by sensing correct shape of WC base pairs -NTP selectivity achieved by multiple contacts between DNAP and minor groove of template base-NTP base pair Adding exonuclease activity --> quick removal of the base analogs
What is the basis of fidelity of RNA synthesis? Discuss kinetic proofreading and structural contributions.
Fidelity achieved by substrate selection and proofreading, isomerization of open to closed active center, exonuclease recognition and removal of mismatched nucleotides Kinetic proofreading -incorporation of incorrect rNTP --> fraying and misalignment of the active site -RNA 3' end no longer in position for inline nucleophilic attack of the phosphodiester bond between the alpha and beta phosphates of the incoming NTP substrate (3' end frays away from the template) -then RNAP pauses and bakctracks, prevents new rNTP from binding -positions for dinucleotide cleavage, then repositions for new inline attack
How do you do nearest-neighbor calculations?
G(formation) = G(initiation) + n*G(AU terminal) + G(symmetry) + sum of neighbors
What highly conserved nucleotides are found at the 5'SS, 3'SS, branch point?
GU at the 5'SS A at the branchpoint AG at the 3'SS
Given its very high reactivity, explain why ribose could not be a component of the first genetic material that gave rise to life.
Genetic material needs to sotre info and pass the info onto progeny, but the reactivity of RNA means it's unstable and would degrade
Outline the experiment used by Meselson and Stahl to distinguish between these three alternate hypotheses (make sure you know about the model system, type of labeling, and type of separation used). Match each competing hypothesis with the results.
Grow E. coli for several generations in 15NH4Cl medium for several generations Transfer to 14N medium and allow replication to occur Density-dependent centrifugation after ~1 generation time -for semi-conservative and dispersive, would see DNA sediment in the middle -for conservative, see two bands (one at top, one at bottom) **at this point, can tell the difference between semi-conservative and dispersive if we denature DNA under high temp conditions (for semi-conservative --> one band at top, one at bottom; for dispersive --> one band in the middle) After 2 generation times -2 bands (one would be in the middle and one would be at the top, corresponding to just 14N)
What is the correct oder of transcriptional events occurring after RNAP binds to the promoter?
Holoenzyme binds loosely to promoter = closed promoter complex Holoenzyme melts DNA at promoter = open promoter complex, pol tightly bound Transcription initiation and promoter clearance Once elongation commences, sigma subunit released and pol leaves the promoter Core polymerase continues to elongate RNA -nucleotides added sequentially -polymerization (5' --> 3') are the core pol moves from the 3' --> 5' direction on the antisense/noncoding strand -pol maintains short melted region of template DNA, while strain is relaxed by topoisomerases (DNA unwound ahead of pol and rewound behind) Proofreading also occurs
What is fidelity?
How well does the enzyme bind correct vs. incorrect nucleotides Once it binds, how fast does the polymerase add the correct vs. incorrect nucleotide do the growing chain Consider the magnitude of kpol/kD for correct vs. incorrect nucleotide
Outline Hoagland and co-workers experiment: hypothesis, experimental approach, results, and conclusion.
Hypothesis: tRNA connects mRNA to protein Approach 1. incubate cytoplasmic RNA of unknown function (that can be labeled with C14 L-amino acids in presence of ATP) + amino-acid activating enzymes + ATP + NTP-generating system 2. purify cytoplasmic RNA-C14 amino acid --> add to ribonucleoprotein particles of microsomes + GTP + NTP-generating system 3. take aliquots of rxn as function of time and centrifuge; collect sup (soluble RNA) and pellet (ribosome) and assay for radioactive label Results: -initially, tRNA has a lot of radioactivity -eventually, the radioactivity of the tRNA goes away but appears in the ribosome fraction! Conclusion: tRNA serves as an immediate carrier of amino acids in protein synthesis
What is the accurate order of events in translation initiation?
IF1 binds A site, helps dissociation of 50S and 30S subunits IF3 binds E site, prevents subunit re-association mRNA binds 16S rRNA of small subunit (30S) -3' end of 16S rRNA (pyrimidine rich) binds Shine-Dalgarno sequence (purine rich) and helps position AUG at P-site fMET-tRNAfMET escorted by IF2 (bound to GTP), base pairs with AUG Conformational change in 30S, release of IF3, 50S associates IF2 hydrolyzes GTP, all IF's dissociate Left with the initiation complex!
Compare and contrast the peptidyltransferase and peptide release reaction mechanisms.
In the peptidyltransferase/transesterification reaction... -a base abstracts a proton from the amino group of aminoacyl-tRNA -then, attack on the carbonyl of the growing peptide chain -leaving group is the tRNA-OH in the P site For peptide release... -Gln185 holds a water molecule in place, while a base abstracts a proton -hydroxide serves as a nucleophile to attack the carbon of the carbonyl --> release of the polypeptide chain with the leaving group being the empty tRNA molecule
Why might an artificially made oligonucleotide containing snRNA sequences inhibit or enhance splicing.
Inhibit splicing The oligos will bind the mRNA, competing with the snRNA's
What happens to maternally encoded DNAP as the zygote's development proceeds?
It is key in early development during DNA replication and cell division High concentrations of maternally encoded DNAP stored in the egg, but these rapidly diminish Quickly, new DNAP enzymes are synthesized from zygotic DNA
What would happen to a zygote's development in the absence of maternally encoded DNAP?
It would not develop - it needs maternal DNAP to replicate its DNA because DNA + 0 = 0 (DNA is insufficient)
What are the key properties of an information molecule? (4) What chemical properties make DNA or RNA an information molecule?
Key properties of an information molecule: stability, inheritable, contain coded info and instructions, must be a template to make copies with minimum error DNA is an antiparallel double helix where the sequence of one strand determines the sequence of the other à suggests a copying mechanism!!
What is the process of accommodation
Large conformational change occurs when the correct tRNA has been brought into the A site --> 3' end of the tRNA switches from pointing away to pointing toward the growing chain The attached AA is now close enough for the peptidyl transferase chemistry to occur
Where is the peptidyl transferase center of the ribosome? Where is the decoding center of the ribosome?
Large subunit Small subunit
How can you identify whether a structure is A-DNA, B-DNA, RNA or DNA-RNA hybrid?
Look for 2'-OH groups (oxygens are normally red) Look at the bases - uracil or thymine Major groove vs. minor groove -the major groove is always deep, while the minor groove is always shallow -for A-DNA, the major groove will be deep but narrow, while the minor groove will be shallow but wide -for B-DNA, the major groove will be deep and wide, while the minor groove will be shallow and narrow
How might mutations on any of the "faces" of DNA or RNA affect function?
May add/remove H-bonding partners and affect the ability of cellular machinery to recognize the DNA
How did Breslauer do his work and what was his general conclusion?
Measured the amount of dsDNA formed by tracking the decrease in UV absorbance to obtain Kd, which gives deltaG Found that base sequence (not composition) determines stability - order of the sequence matters!! -e.g., AAGG would not be same as AGAG
What is an example of an OFF/ON switch?
Most riboswitches undergo conformational change upon binding cognate effector molecule --> genetic off/on switch
How do you perform nearest neighbor calculations for hairpin structures?
Need to add penalty for the bulge + penalty for dangling residue + GNRA bonus + sum of nearest neighbor interactions
Based on the table from genome transplantation into M. capricolum, what conclusion can be drawn from the number of colonies that grew in the negative controls? No donor DNA: no colonies, except one trial that had 2 colonies No recipient cells: no colonies
No donor DNA: DNA is necessary (the 2 colonies were contamination) No recipient cells: DNA is not sufficient
Where does splicing occur?
Nucleus
Outline the experiment used by Okazaki to distinguish between continuous, semi-discontinuous, or discontinuous replication.
Observed E. coli with ts ligase For WT, as time goes on, we see fragments appearing that are farther from the top of the tube (after density-dependent centrifugation) --> they are getting larger For mutants at 43 degrees -lot of small fragments no matter how much time progresses -but, there are always fragments that are large as well -therefore, semi-discontinuous replication Finally, if you go from 43 to 30 degrees, size of fragments gets larger -thus, the ligase is the enzyme that is responsible for stitching the fragments together
What is kinetic proofreading?
Occurs in multiple steps, rates are fine-tuned to mamximize the speed of correct reactions while slowing down and reversing incorrect reactions
True or False: a. 2'-OH group has made RNA more stable for info storage b. 2'-H group has made RNA more stable for info storage c. 2'-H group has made DNA more stable for info storage d. 2'-OH group has made DNA more stable for info storage
Only c is true
True or false: a. only one of the three active sites can be occupied by a tRNA molecule at any given time b. each tRNA that associates with the ribosome must first bind in the P site and then move to the A site c. the P site contains the tRNA molecule covalently bound to the growing chain of amino acids d. all tRNAs leave the ribosome through the A site
Only c is true
The portion of the ribosome that specifically recognizes the bacterial Shine-Dalgarno mRNA sequence is: (true or false) a. protein S5 of the 30S subunit b. N-formylmethionine-tRNA c. 23S rRNA of the 50S subunit d. 16S rRNA of the 30S subunit
Only d is true
True or False: a. ribosomal translocation occurs before the first aminoacyl-tRNA molecule binds to the small subunit b. initiation of translation occurs only after the small subunit dissociates from the mRNA c. the large subunit of the ribosome binds the mRNA before the small subunit d. the first aminoacyl-tRNA molecule binds to the small subunit before the large subunit binds
Only d is true
Explain functional mimicry of tRNAs and release factors indicating what specific structural motifs are employed by the release factors to mimic specific tRNA structural features.
PVT tripeptide matches tRNA anticodon arm -uses side chains to recognize the stop codon GGQ matches with the CCA end of the tRNA (the amino acid arm) Overall, RF1 mimics tRNA and competes in the A site
Describe the kinetic contributions to fidelity during translation.
Peptidyl-tRNA selection -all aminoacyl-tRNAs escorted into A site to sample codon-anticodon interactions; initial binding/sampling is similar for all -codon recognition: dissociation occurs faster for non-cognate -GTPase activation: fast for cognate, slow for non-cognate and near-cognate Proofreading -GTP hydrolysis allows for accommodation: accommodation fast for correct, rejection fast for incorrect
What are some medical consequences of mis-splicing? (6)
Prader Willi syndrome, spinal muscular atrophy (SMA), cancer, frontotemporal dementia, CF, ducehnne muscular dystrophy (DMD)
What are the components of the prokaryotic and eukaryotic ribosomes?
Prokaryotic (70S) -Small subunit (30S) = 16S rRNA + 21 proteins -Large subunit (50S) = 5S rRNA + 23S rRNA + 31 proteins Eukaryotic (80S) -Small subunit (40S) = 18S rRNA + 33 proteins -Large subunit (60S) = 5S rRNA + 5.8S rRNA + 28S rRNA + 50 proteins
Watson and Crick couldn't fit purine-purine and pyrimidine-pyrimidine in their model. Why do purines base pair with pyrimidines, but not purine-purine nor pyrimidine-pyrimidine?
Pyrimidine bases are slightly smaller, while purines are larger; if we get base pairing between pyrimidine-pyrimidine or purine-purine, this would not fit into the double helix because the diameter of the helix across these base pairs would either be too small or too large.
Describe the kinetic partitioning of the DNAP enzyme.
REversible nucleotide binding occurs for all 8 nucleotides Once correct nucleotide binds, active site closes very quickly -for correct nucleotide, forward rate is VERY large, while reverse is VERY slow Then, elongation chemistry occurs and enzyme opens again Finally, translocate to next base and release of PPi
What stop codons do each release factor bind?
RF1 binds UAA and UAG RF2 binds UAA and UGA
Where do replication and transcription begin? (i.e. at what point in the DNA)
Replication begins at an origin of replication Transcription begins at promoters -there are -35 and -10 consensus sequences that RNAP can recognize and allow for binding
Name cellular contexts for nearest neighbor analysis.
Replication: DNA melting, DNA/RNA hybridization Transcription: DNA melting, DNA/RNA hybridization Translation: codon-anticodon recognition Splicing: RNA/RNA pairing RNA signaling, regulation (e.g., ribozymes, microRNA, riboswitches)
What are the three components of monomer units that the cell uses for making the building blocks to synthesize nucleic acids?
Ribose or deoxyribose (D-furanose sugar) -beta configuration (OH at the anomeric carbon is pointed up) Phosphate Nitrogenous bases (AG = purines, CTU = pyrimidines)
glmS riboswitch: Where is it found? Be specific - what gene and where in that gene? What metabolite does it bind? What happens when it binds its ligand? What is known about the active site?
Ribozyme Part of the mRNA encoding the GlcN6P synthetase enzyme -resides in the 5' UTR of the glmS gene Controls production of the metabolite it binds (GlcN6P) When GlcN6P binds --> self-cleavage --> degradation of the mRNA -during the cleavage, a base abstracts a proton from the 2'-OH of A(-1); attacks the phosphorous linking A(-1) and G1; breaking of the phospohdiester bond between A(-1) and G1 Active site requires water and Mg (it is a metalloenzyme); subtle changes w/in the active site --> riboswitch can't bind its ligand
What is the mechanism of gropu II self-splicing introns?
Same as the spliceosome The 2'-OH of the branchpoint A serves as the nucleophile that attacks the 5'SS Then, the 3'-OH of the 5' exon attacks the 3'SS Result is the 5' exon joined to the 3' exon The intron has been excised but is in a loop
DNAP has ____________ and _____________ capabilities. -Describe these capabilities.
Selectivity and proofreading Selectivity: When we incorporate the right nucleotide, the active site closes but if we have the wrong nucleotide, the rate of closure of the active site is very slow Proofreading: -If the wrong nucleotide gets incorporated, then the rate of polymerization for incorporating the next nucleotide is very slow -For wrong nucleotide, the rate for transferring to the exonuclease domain increases
What are microRNAs? How do they work? What is their function?
Single-stranded, 22nt, noncoding RNA Can base pair to sequences w/in the 3' UTR of an mRNA (the 3' UTR is known to have sites for proteins to bind that are important for regulation) Function in post-transcriptional regulation of gene expression Also, microRNAs found in intronic regions can compete w/alternative splicing
In a couple sentences: how does the ribosome recognize cognate from near cognate
Small subunit is the decoding center - the 16S rRNA can interact and sense the shape of the codon-anticodon interaction Near cognate loses interactions with the minor groove Feet of the tRNA (anticodon) tell the arm what to do - for near cognate, the tRNA doesn't fit into the ribosome
How is the bacterial Shine-Dalgarno mRNA sequence specifically recognized?
Small subunit of ribosome is the "decoding center" - 16S rRNA (part of the small subunit) pairs with the Shine-Dalgarno sequence to help position the AUG codon into the P-site of the ribosome The 3'-end of the 16S rRNA pairs with the Shine-Dalgarno sequence in the 5'-end of the mRNA Specifics: Shine-Dalgarno sequence is a purine rich region which hydrogen bonds to the pyrimidine-rich 3'-end of the 16S rRNA
Describe how microRNAs can compete w/alternative splicing.
Some microRNAs are found in intronic regions Can offer alternative splice sites!
In vivo [rNTP] >> [dNTP]. Explain how the cell prevents incorporation of dNTP over rNTP during replication.
Steric gate formed by two amino acid side chains in the active site that sandwich the substrate sugar moiety to exclude a 2'-OH group using sterics and electrostatic repulsion Y12 and Y115 do not like the 2'-OH group If we mutate Y115A, then rNTPs can get incorporated much more easily
Describe the events of translation termination.
Stop codons recognized by class I release factors (RFs) RF binds in A site --> allows water into active site Completed polypeptide released Ribosome dissociates into its two subunits
What are riboswitches? Give examples discussed in class (don't need to give the mechanism)
Structured domains of UTRs and introns of mRNAs that can recognize small molecule metabolites and second messengers and regulate gene expression in cis; some can also undergo ligand-induced self-cleavage or hybridization in trans Noncoding RNA that can detect the concentration of various ligands in the cell --> conformational changes depending on cellular conditions --> dictates function GlmS SAM-II TPP
Compare and contrast the Syn1.0 and 3.0 cells made by Venter's group in regards to cell size and morphology and doubling time. Based on Venter's Science article, what are the limitations of using "stripped down" genomes to "reboot artificial life"? For example, further research indicated they needed to include 3 "pseudogenes" and 70 proteins of unknown function as part of 130 functionally uncharacterized proteins. What are the implications for synthesizing new life forms?
Syn3.0 grew smaller and 3x slower (longer doubling time), heterogeneous morphology Clearly the Syn3.0 cells had the genes necessary for survival but were missing some that were important for optimal growth Limitations -you don't necessarily know what genes need to be included Implications -there are still many genes/proteins that are important for life that we don't know the function of yet
What is the mechanism of group I self-splicing introns?
The 3'-OH of an exogenous G serves as the nucleophile that attacks the 5'SS Then, the 3'-OH of the 5' exon attacks the 3'SS Result is the 5' exon joined to the 3' exon The intron has been excised and has the exogenous G at its 5' end
The X-ray fiber diffraction pattern was reported by Wilkins (third group to report structure of DNA in the same issue of 1953 Nature journal) to be the same for all sources of DNA ranging from viruses to mammals. In one sentence, speculate on what this observation implies about the overall 3D structure of DNA from all organisms.
The 3D structure of DNA is universal across all organisms - i.e. it is the same!
What is the difference between the sequence hypothesis and the central dogma?
The central dogma is a negative statement that says that we cannot start with a protein and then synthesize the corresponding nucleic acid; the flow of information between nucleic acid and protein is unidirectional The sequence hypothesis is what people normally think of as the central dogma - it is the general flow of information from DNA --> RNA --> protein (positive statement)
How do individual aminoacyl-tRNA synthetase enzymes recognize the correct tRNA molecules through the anticodon sequence? How to they recognize the correct amino acid?
The enzyme can recognize the correct tRNA by interacting with the anticodon sequence via the anticodon binding domain and through other features of the tRNA structure -Different tRNAs have slightly different overall configurations which can be recognized by the appropriate aminoacyl-tRNA synthetase When the incorrect amino acid gets added to the tRNA, it gets shunted to the editing domain where it can be hydrolyzed
What is one key difference between group I and group II introns splicing mechanisms?
The nucleophile for group I introns is the 3'-OH of an exogenous G, while for group II, it is the 2'-OH of the A (branch point)
Name 2 features of the Pauling and Corey X-ray structure that was wrong according to Watson and Crick. List 2 arguments used to arrive at this conclusion.
The phosphates are on the inside -The phosphate groups are in the salt form (don't have the acidic hydrogens); the negatively charged phosphate groups on the inside would repel each other and there would be nothing holding the strands together -Some of the van der Waals distances are too small to be consistent with phosphates on the inside There are three intertwined chains -Didn't indicate reasons why a triple helix were wrong; rather, they assumed the double helix and detailed how the two chains are held together by hydrogen bonding between a pyrimidine and a purine
Discuss the thermodynamics of DNA duplex formation and denaturation.
Tm is a function of sequence composition, salt, strand concentration, and modifications Tm is the temperature at which 50% is double-stranded, while 50% is single-stranded GC-rich or high salt conditions both increase Tm -GC base pairs have 3 H bonds -salt stabilizes the double helix bc it neutrlaizes negative charge on the phosphates
There are 5 snRNAs that are coupled to proteins to form snRNPs that make up the spliceosome (U1, U2, U4, U5, U6). What are their functions
U1 - binds 5'SS U2 - binds branch site, part of catalytic center U4 - masks catalytic activity of U6 U5 - binds 5'SS and 3'SS U6 - catalyzes splicing
How is fidelity/specificity achieved for the spliceosome? Specifically, make sure to mention Prp5, Prp43, Prp16, and Prp22
U2 snRNP binds pre-mRNA -Prp5-dependent rejection competes with branch site recognition by U2 -Prp5 activity then stabilizes U2 association w/substrate Addition of U4/U6/U5 snRNP leads to U1 and U4 release and formation of catalytically active ribosome -Prp16-dependent rejection competes with 5'SS cleavage -if rejection occurs, Prp43 promotes dissociation of spliceosome After 5'SS cleavage, Prp16 promotes exon ligation conformation of the spliceosome -Prp22 helps with rejection, Prp43 promotes dissociation of spliceosome Exon ligation and release of nascent mRNA from spliceosome (mediated by Prp22) Then, Prp43 promotes release of excised intron and dissociation of spliceosome
What are the three stop codons?
UAA, UAG, UGA
Describe the experiment Venter performed with the M. capricolum and M. mycoides cells. What did he prove? What happened with the antibody plates?
Used MC cells with DNA removed, used gDNA from MM cells (MM DNA tagged w/LacZ and tetracyclin resistance genes) MC cells w/o DNA --> no colonies (negative control: DNA is necessary) Just donor MM gDNA --> no colonies (negative control: DNA is not sufficient) MC cells + buffer --> no colonies MM cells + MM gDNA --> blue colonies with MM phenotype For MC WT cells, MC antibodies bind but MM don't For MM donor cells, MM antibodies bind but MC don't For transplant cells (MC cells + MM gDNA), the MM antibodies bind but the MC don't -successfully transformed the phenotype of the cell!
Identify the grooves/faces of DNA. What effect would mutations in any of the faces have? What is pseudouridine?
Watson-Crick edge is the H-bonding surface -mutations would prevent correct base pairing (methylation would remove an H-bonding opportunity) Hoogsteen edge = major groove face -methylation on Hoogsteen edge can affect recognition machinery Sugar edge = minor groove face Pseudouridine results from the actions of pseudouridine synthetase -binds and severs the C1'-N1 bond -rotates uracil base about the C6-N3 axis -adds an H-bonding partner to the Hoogsteen/major groove face
What was the conclusion from Venter's paper about chemically synthesizing DNA? What didn't work at first? After the publication of 2010 Science paper, ABC news carried this headline "Scientists Create First 'Synthetic' Cells". Is this a scientifically accurate statement? Yes/No? Explain your answer.
We can chemically synthesize DNA and put it into a reicipient cell and boot it up -used chemo-enzymatic synthesis to assemble the M. genitalium genome (chemical synthesis of 60 nt fragments, stiched fragments together w/enzymes, then larger fragments assembled in E. coli and then yeast) -transplanting chemically synthesized DNA to cells was successful At first, transplant of the DNA to a new cell didn't work because they had synthesized the bacterial chromosome in yeast (yeast do not methylate DNA, but bacteria does) -the host bacterial cell was degrading the DNA because it wasn't methylated -fixed the issue by removing the restriction enzymes from the recipient cells Not exaclty accurate -needed chemoenzymatic synthesis -also, needed to transplant the DNA into a prexisting cell - DNA lone isn't sufficient to create a synthetic cell
Explain how DNAP active site aligns correct dNTPs for catalysis? Be specific - use specific structural elements of DNAP and the structure of the active site. Answer these questions in regards to DNAP: -what happens when the correct dNTP is incoporated (reference a specific structural element) -why is the above step important? -how can DNAP sense incorrect base pairings?
When correct dNTP incorporated, the finger domain's O helix undergoes a conformational change that closese the active site around the correct dNTP, holding it in place Closure is important for catalysis -helps align the Mg2+ ions and the 3'-OH for inline attack on the alpha phosphate DNAP can sense incorrect base pairings because of different C1'-C1' distances and glycosidic angles
Discuss Avery's work/experiments that helped prove DNA is genetic material. -use of viscosity -scoring assay -griffith experiments
Worked to isolate the "transforming principle" from crude bacterial extract -tested viscosity as marker for intact DNA (when DNAse added, viscosity of the transforming principle was lost) -note that if a combination of the "transforming principle" + DNAse is heated, the DNAse is denatured --> intact DNA --> high viscosity Used scoring assay - smooth/large colonies (virulent) or rough/small colonies (avirulent) -add heat-killed DNA from S strain to avirulent R strain bacteria -found diffuse growth and SIII colonies form if enough heat-killed DNA from S strain is added Repeated the Griffith experiment -took extract from heat-killed virulsent S strain -treated it with DNAse, RNAse, or protease -only DNAse treated extract couldn't transform avirulent to virulent pneumococcus
What does it mean when deltaG is on the order of kT (thermal energy)?
You can't form the bond - thermal energy is enough to break the bond deltaG >> kT for a bond to form
This stage of translation is the most important to the fidelity of protein synthesis (pick one) a. formation of aminoacyl-tRNAs b. initiation of translation at an AUG codon c. translocation of the peptidyl-tRNA from the A site to the P site during elongation d. termination of translation at a stop codon
a
True or false: a. protein synthesis begins with the N-terminal end b. the 5'-AUG initiation codon specifies an N-terminal methionine residue c. the ribosome is degraded after dissociating from the stop codon
a is true b I said false because it's technically a formyl-methionine (he accepted this as the case, but probably gave credit for people who said true) c is false
Define the A, P and E sites of the ribosome
a. A = aminoacyl site; binds incoming aminoacyl tRNA b. P = peptidyl transferase site; holds the tRNA with the growing polypeptide chain i. Note that the AUG is positioned into the P site and fMet-tRNAfMetis the only tRNA that initially binds here c. E = exit site; serves as a threshold - holds the tRNA w/o its amino acid --> then gets released by the ribosome
Describe the experimental evidence for RNA primers at the beginning of Okazaki fragments. Explain the results using the general mechanism of nucleic acid hydrolysis by internal 2'-OH.
a. Induce base-catalyzed hydrolysis of RNA (at high pH) - add NaOH b. If we have 5' RNA primer, we expect our nucleic acid fragment length to partially degrade/shorten i. Also, the functional group at the end of the DNA would be a 5' hydroxyl (instead of the normal phosphate) c. In the actual experiment, we can label the presence of the 5'-OH on the DNA with [γ32P]ATP using a T4 polynucleotide kinase i. Any DNA-only fragments will remain unlabeled
What are some uses of nearest neighbor analysis (5)?
a. Predict stability of probe-gene complex b. Select optimal conditions for hybridization experiment c. Deciding on minimum length of a probe d. Predict influence of specific transversion or transition on stability of affected DNA region e. Predict regions of local melting
What are some cellular uses for cyclic or dicyclic nucleotides?
cAMP is a second messenger Cyclic dinucleotides -motility -biofilm formation -virulence -bacterial sporulation -reports DNA damage -linked to FA synthesis -antibiotic resistance -cell wall homeostasis -promotes intestinal colonization of human host in V. cholerae
How are deltaG, Kd, and Ka related?
delta(G) = RTln(Kd) Kd = 1/Ka
Define cognate, near-cognate, and non-cognate
i. Cognate = all 3 base pairs match ii. Near-cognate = almost same codon, but 1 mismatch iii. Non-cognate = incorrect
What were the three competing hypotheses about DNA replication before the Meselson-Stahl experiments?
i. Semi-conservative = parental strands separate, each serves as a template to make a new complementary strand ii. Dispersive = DNA breaks apart into fragments and each fragment serves as a new template for a new DNA fragment - every cell division produces two cells w/varying amounts of old and new DNA iii. Conservative = entire DNA double helix serves as a template for a new helix
What is processivity?
kpol/koff How fast can I keep making DNA without DNAP falling off?
What recognizes the 5'SS, 3'SS, and branch point?
snRNPs! 5'SS recognized by U1 (binds in ATP-independent manneR) BP recognized by U2 (needs ATP) 3'SS reocgnized by U5