Genetics 467 Exam 4
enzymes needed for semidiscontinuous DNA replication
DNAP = synthesizes DNA helicase = unwinds DNA strands primase = synthesizes RNA primer DNAP (again) = fills in gaps left by primers ligase = connects Okazaki fragments topoisomerases = remove supercoils
Which of the following statements about the genetic code is also FALSE?
For half of the codons, mutating the third position to any other nucleotide will not change the amino acid Codons for polar/negatively charged amino acids only differ at the third position Codons for polar/positively charged amino acids have a purine at the second position 16 codons begin with a U *Every codon can be mutated to a stop codon by a single nucleotide mutation*
what replicates COVID-19?
RNA-dependent polymerase (RdRp). synthesizes in 5' -> 3' direction without primer, copies +gRNA to make -gRNA which then serves as a template for +gRNA, and the cycle continues
ribosomes
RNA/protein complexes
Do the following polymerases need a primer?
Taq DNAP used for PCR = yes DNAP in DNA replication = yes bacterial RNAP = no primase = no RNAP 1 = no RNAP 2 = no RNAP 3 = no
Which of the following is the most common type of DNA base pair?
Tautomeric base pair Ionized base pair Wobble base pair *Watson-Crick base pair* Mismatch base pair
How does the Johnson and Johnson vaccine work?
1. spike protein is purified 2. adenoviral vector is injected 3. body produces spike protein 4. immune system makes antibody involves protein, viral vector, and mRNA methods
The reaction of EMS with guanine generates O-6-ethylguanine, which lead to a G-C to A-T transition. Write out the DNA replication steps that lead to the transition.
5'---G---3' 3'---C---5' (EMS) 5'---G+O6 ethyl---3' 3'---------C--------5' (DNA replication) 5'---G+O6 ethyl---3' 3'-------T----------5' and 5'---G---3' 3'---C---5' (DNA replication) 5'---G+O6 ethyl---3' 3'-------T----------5' and 5'---A---3' 3'---T---3' and 5'---G---3' 3'---C---5' and 5'---G---3' 3'---C---5'
Which AUG codon is in the best context for translation initiation in eukaryotes?
5'GGUUUAUGU3' 5'GGCUUAUGU3' 5'GGGUUAUGU3' 5'GGGUUAUGC3' *5'GGAUUAUGG3'* (Kozak)
All of the following factors interact with the tRNA acceptor stem EXCEPT:
Aminoacyl-tRNA synthetases Large ribosomal subunit EF-Tu *mRNA codons* eEF1a
amorph
complete loss of function. no gene activity encoded. usually nonsense, frameshift, or nonconservative missense
how is translation regulated?
developmental and environmental signals turn it on and off. miRNAs also regulate by base pairing to specific mRNAs, and turning off translation that way, which can lead to cap binding (eIF), subunit joining, circularization, elongation (eEF), proteolysis, miRNA or RNA-BP activities, termination (eRF) or initiation
SARS-Cov-2
made up of the following: nucleocapsid that binds genome and forms long helical RNP structure, spike that binds host receptor ACE2 on lung/heart/kidney cells, envelope for virus assembly/envelope formation/budding, membrane for viral integration, ssRNA genome (30000 nt, encodes about 30 proteins)
hypomorph
partial loss of function. less gene activity than wild type. usually missense, can be in regulatory region
what recycles ribosomes?
eIFs, ABCE1, ligatin
how are telomeres not recognized as a break in DNA's 2x strand?
exposed telomere end -> TRF1 dimer pairs to duplex DNA -> TRF2 dimer invades 3' overhang -> t-loop created, which prevents 2x strand break
specific tRNA-fMet involved in initiation?
fMet = formyl methionine
Fen1
flap endonuclease
Ames test
identifies compounds that induce mutations. see if chemicals are mutagenic/carcinogenic. spontaneous = his+ revertant colonies were sparse. induced = his+ colonies everywhere, sign of mutagenic compound ex: EtBr as carcinogenic...replaced with SYBR green 1
example of in-frame insertion?
in Huntingtion gene, lead to disease. repeat CAG sequence codes for glutamine...in normal person, 6-35 are present. in Huntington's patient, have more than this amount on an increasing level of severity with each addition
what's true of miRNA's role in development?
it has important ones ex: gain of function experiment with overexpression of single miRNAs in bug wings leads to whacky phenotypical changes
what's true of each type of DNA damage?
it has its own corresponding repair mechanism
what's true about capping of +gRNA and +sgRNA?
it involves SARS-CoV-2 enzymes...RNA triphosphotase, N7 methyltransferase (MT), 2'-O methyltransferase, guanylyl transferase (GT),
what's true of protein localization?
it is altered by post-translational modifications. classic nuclear localization signals can transport signals upregulated by phosphorylation, or downregulated by phosphorylation
what does base pairing of tRNAs to rRNAs do?
it positions the tRNAs at the peptidyl-transferase center, which is rRNA. at the A site, base pairs to CCA of tRNA, and rRNA at P site base pairs to CCA of tRNA too
where does DNA replication start, and where does it move?
it starts at the origins, and spreads in both directions. bacteria have only 1 origin for replication, eukaryotes have many. at the origin, leading and lagging strands switch
what do replication mechanisms do?
reduce # mutations. DNAPs have proofreading activity...detect incorrect base and remove it (3' -> 5' endonuclease activity) by process of extension (incorrect A to C) proofs (detect and remove) and extension again (correct G added)
what's required for synthesis of COVID-19?
translation frameshift. polyproteins then get cleaved by proteases that are made by genes 3 and 5
when is translation terminated?
when release factors bind to stop codons. in bacteria, RF1 recognizes UAA/UAG and RF2 recognizes UAA/UGA. in eukaryotes, eRF1 does the jobs of RF1 and 2, and eRF3 does RF3's job
bacterial ribosomes
whole thing = 70S big subunit = 50S little subunit = 30S
eukaryotic ribosomes
whole thing = 80S big subunit = 60S little subunit = 40S
why are telomeres/telomerases biologically important?
with age, somatic cells' telomeres get shorter and shorter, and telomerase becomes inactive one the Hayflick limit is reached and the telomeric buffer is depleted. however in stem cells, sperm, cancer cells, and eggs, telomerase remains active throughout life.
1Which of the following statements about aminoacyl-tRNAs is FALSE?
They are also called charged tRNAs They contain an amino acid linked to the 3' end of a tRNA *They are sometimes bound in the E site of ribosomes* They are generated by aminoacyl-tRNA synthetases They are substrates for both translation initiation and elongation
what does polyploidy as a result of endoreplication often do?
confer resistance to environmental stresses not tolerated by diploid cells ex: increasing # polyploid brain cells in flies as they age to protect against neurodegenerative diseases correlated with DNA-damage induced death
translesion synthesis (S)
depurination (non-bulky damage), pyrimidine dimer, bulky adduct
direct repair (G1)
alkylation (non-bulky damage) or pyrimidine dimer
base excision repair (G1)
alkylation, oxidation, deamination, depurination...non-bulky damage
wobble base pairing
allows tRNA to recognize 2 or more codons, as the same anticodon at the tRNA loop can give different codons. as few as 31 tRNAs can read all 61 amino acid codons. ex: AGG anticodon, get UCC/UCU which are both serine due to C-G or C-U base pair. A to I editing extends wobble base pairing of tRNAs *NOT mRNAs* 5' end anticodon 3' end codon A U C G G C/U U A/G I A, C, or U
where do interacting factors work for tRNAs?
amino acid attachment site and acceptor stem: aminoacyl-tRNA synthetase, large ribosomal subunit, EF-Tu and eEF1a variable T/C loop and D loop: large ribosomal subunit anticodon stem, anticodon loop: aminoacyl-tRNA synthetase, small ribosomal subunit, mRNA codons
what do all amino acids have?
amino group, carboxyl group, R group, and a side chain
How to predict what sequence will be encoded by mRNA?
anything before AUG = 5' UTR anything after stop codon UAA, UGA, UAG = 3' UTR
How many different codons make Val, Pro, Thr, Ala, and Gly?
4
3 stop codons
UAA, UAG, UGA
What codons can be mutated to generate a UGA nonsense mutation?
UAG stop: 1st codon of Lys (AAG), Gln (CAG), Glu (GAG) 2nd codon of Ser (UCG), Trp (UGG), Leu (UUG) 3rd codon of Stop (UAA), Tyr (UAC or UAU) UGA stop: 1st codon of Arg (CGA or AGA), Gly (GGA) 2nd codon of Leu (UUA), Ser (UCA), Stop (UAA) 3rd codon of Cys (UGU or UGC), Trp (UGG)
What single nucleotide mutations in the UCA codon would produce silent (synonymous) mutations, conservative (nonsynonymous) missense mutations, nonconservative (nonsynonymous) missense mutations, and nonsense mutations?
UCA = Serine silent mutation = (UCU-ser, UCC-ser, UCG-ser) conservative missense mutation (ACA-thr) nonconservative missense mutation (CCA-pro, GCA-ala, UUA-leu) nonsense mutation (UAA-stop, UGA-stop)
what're examples of consequences of mutant DNA repair mechanisms?
Xeroderma pigmentation due to inhibited NER. 1/100000, causes premature skin aging and increased risk of cancer due to an inability to repair DNA damage from UV light. mutations are in genes encoding 7 nucleotide excision repair proteins Ataxia-Telangiectasia (A-T) caused by mutation in ATM genes. 1/40000, ATM protein is important so has sweeping effects throughout body and results in multisystem disorder and neurodegeneration. mutations are scattered throughout gene
Could the spacing of origins in eukaryotic organisms affect the amount of time it takes to replicate a chromosome? Explain your answer.
Yes, spacing can affect the amount of time it takes to replicate a chromosome. The fastest replication would occur if origins were equally spaced along a chromosome, and the slowest replication would occur if origins were tightly clustered. For example, presume that there are 5 origins for a 1 Mb chromosome and the rate of replication was 2 kb/min. As shown in the diagram below, equally spaced origins would take 50 min to replicate the chromosome (remember that replication is bidirectional, so each of the 5 origins would replicate 200 kb in 50 minutes to total 1 Mb). In contrast, clustered origins would require some origins to replicate more DNA than other origins. In the example in the diagram, one of the replication forks for the origin on the far right would need to replicate 750 kb, so this would take 375 minutes.
how does E3 ubiquitin ligase provide substrate specificity?
a linkage is formed between E1 cysteine and carboxyl terminal glycine of ubiquitin, ubiquitin is transferred from E1 to E2, E2 associates with E3, and ubiquitin is transferred from E2 to E3 cysteine and then the substrate
Which of the following definitions is also INCORRECT?
*Antimorphic mutations are loss-of-function mutations* Amorphic mutations are complete loss-of-function mutations Hypomorphic mutations are partial loss-of-function mutations Hypermorphic mutations are gain-of-function mutations Neomorphic mutations are gain-of-function mutations
Which of the following statements about tRNAs is FALSE?
*At least 61 tRNAs are needed to read all of the amino acid codons* There is one aminoacyl tRNA synthetase for each amino acid tRNAs are charged with an amino acid at the 3' end Wobble base pairing allows tRNAs to recognize more than one codon tRNA anticodons base pair to mRNA codons in the small ribosomal subunit
Post-translational modifications affect all of the following molecular events EXCEPT:
*Base pairing between RNAs* Localization of proteins Interactions between proteins Binding of proteins to DNA and RNA Enzymatic activity of proteins
Which of the following statements about DNA damage is FALSE?
*DNA damage occurs to bases but not to the sugar-phosphate backbone of DNA* Depurination involves base removal by cleavage of the glycosidic bond Water and reactive oxygen species can cause DNA damage Deamination leads to base transitions DNA damage can occur in both coding and non-coding regions of genes
All of the following events occur during translation elongation in bacteria EXCEPT:
*Decoding of AUG codons by fMet-tRNAfMET* Delivery of the aminoacyl-tRNA to the ribosome A site Peptide bond formation Translocation of deacylated tRNAs from the ribosome P to E site Translocation of peptidyl-tRNAs from the ribosome A to P site
Which of the following statements about the cell cycle in eukaryotes is also FALSE?
*In endoreduplication, DNA replication occurs more than once per cell cycle* A typical cell cycle contains G1, S, G2, and M phases Endomitosis is an atypical cell cycle where cells enter mitosis but do not divide Endocycle is an atypical cell cycle that does not contain G2 and M phases Endoreduplication produces polyploid cells
Which of the following factors IS NOT used by both SARS-CoV-2 and eukaryotes for genome replication?
*Ligase* 3' to 5' exonuclease Helicase Single-strand binding protein Processivity factor
DNA replication in both bacteria and eukaryotes involves all of the following EXCEPT?
*Multiple origins of replication* DNA polymerases RNA polymerases Helicases Topoisomerases
Which of the following statements about RNA-dependent RNA polymerases (RdRp) is FALSE?
*RdRps are involved in replication but not transcription of the viral genome* RdRp uses RNA template for the synthesis of RNA RdRp synthesizes RNA in the 5' to 3' direction RdRp does not require a primer to begin RNA synthesis RdRp uses ATP, CTP, GTP, and UTP as substrates for RNA synthesis
All of the following events occur during translation initiation in eukaryotes EXCEPT:
*Ribosome translocation by eEF2* Binding of eIF4E to the mRNA m7G cap Binding of eIF4G to PABP to circularize the mRNA Unwinding of double-stranded RNA by eIF4A Delivery of Met-tRNAiMet to the ribosome P site by eIF2-GTP
All of the following events occurs during translation initiation in bacteria EXCEPT:
*Scanning of the 5' UTR for the AUG codon in the right context* Binding of IF3 and IF1 in the E and A sites to block assembly of the large ribosomal subunit Delivery of fMet-tRNAfMet to the ribosome P site by IF2-GTP Hydrolysis of GTP to release IF2 and assemble the large ribosomal subunit Assembly of a ternary complex containing IF2-GTP and fMet-tRNA^fMet
DNA replication involves all of the following enzymatic activities EXCEPT:
*Synthesis of DNA by beta-clamp* Removal of DNA supercoils by topoisomerase Unwinding of DNA by helicase Ligation of Okazaki fragments by DNA ligase Synthesis of RNA primers by Primase
Which of the following statements about codons is FALSE?
*There are different methionine codons for translation initiation and elongation* During translation, codons are continuous During translation, codons are nonoverlapping There are 61 amino acid codons There are 3 stop codons
nonhomologous end joining repair of DNA 2x strand breaks
*error prone* bacteria: detection by Ku, ligation by LigD/Lig4 eukaryotes: Ku70-Ku80 binds DNA ends once detected, DNA-PKcs binds to Ku70-Ku80 and recruits Artemis for excision, works alongside DNAP to process ends of DNA, XRCC4/XLF/DNA ligase 4 complex ligates processed ends
m7G cap and poly A tail binding proteins?
+ mRNA and eIF-4F binds m7G cap @ 5' cap, eIF4E is bound to cap, and eIF-4A is bound to eIF4G PA13P (PABP?) binds eIF4G @ cap, circularizing mRNA
what do sequences surrounding AUG contribute to initiator codon selection?
-3 and +4 positions before/after AUG are important (Kozak). G plays a role in determining AUG, with A > G. you can regulate the amount of protein made by mRNA by changing nucleoties around AUG, but Kozak sequence maximizes productivity
How many codons make Met and Trp?
1
what's the minimum number of nucleotides needed to uniquely code for 20 amino acids?
1 n = 4^1 = 4 combos 2 n = 4^2 = 16 combos *3 n = 4^3 = 64 combos*
what ensures that translation is accurate?
1. 2x sieve mechanism of aminoacyl-tRNA synthetases ensures the correct pairing of aminos and tRNAs 2. base pairing between mRNA codons and tRNA anticodons leads to the correct amino getting to the A site 3. selection of the start site AUG leads to the correct reading frame (bacteria: Shine-Delgarno and 16S, eukaryotes: ribosome scanning) 4. activation of GTP hydrolysis into GDP ensures that one step in translation is correct before moving on to next step
how do bacteria join Okazaki fragments?
1. DNAP 3 moves along fragment 1 until hits primer 2. DNAP 1 binds to DNA, uses 5'->3' exonuclease activity to eat primer then fills gap 3. DNA ligase links fragments
how do eukaryotes join Okazaki fragments?
1. DNAP delta moves primer, makes flap 2. Fen1 cuts off flap, DNAP delta fills gap 3. DNA ligase links fragments
what 3 enzymes are needed for ubiquitination?
1. E1 ubiquitin activating enzyme (1/genome) 2. E2 ubiquitin conjugating enzyme (more/genome) 3. E3 ubiquitin ligase (most/genome...~600 in humans) which specifically provides substrate specificity for ubiquitination
2 proofreading steps of aminoacyl-tRNA synthetase?
1. activation site makes it so that larger amino acids are rejected 2. editing site makes it so that smaller amino acids get hydrolyzed
4 consequences of post-translational modifications?
1. altered interactions...phosphorylation allows kinase to interact with adapter/adaptor protein 2. changed localization...phosphorylation makes it so that kinase moves from cytoplasm to nucleus 3. changed function...phosphorylation changes DNA binding or protein binding of transcription factors 4. changed stability...phosphorylation changes the half life of proteins
COVID-19 replication cycle
1. attachment and entry, spike protein to ACE2 receptor 2. uncoating to expose +gRNA 3. translation of +gRNA to make polyproteins 4. proteolysis involving proteases and RNA-dependent RNAP 5a. replication involving replicase and RTC 5b. transcription of sub-genomic RNAs 6. translation of +sgRNAs into M, E, S proteins 7. assembly of virus
5 steps to DNA repair
1. detection of bad DNA 2. incision of bad DNA 3. excision of bad DNA 4. synthesis of good DNA using undamaged DNA as template 5. ligation of new/old DNA strands *not all occur with every repair mechanism*
steps to termination of DNA replication?
1. fork convergence via TopoIV and 3' flap generation 2. synthesis completion (helicase dissociation, 3' flap removal by exonuclease, gap filling by DNAP 1) 3. replisome assembly (Okazaki maturation) 4. ligation and decatenation (by topoisomerase)
how do miRNAs inhibit mRNA translation and promote decay?
1. mRNA being translated in closed loop, add GW182 protein and AGO protein to make miRISC that binds to mRNA 2. binds to region of pre-poly A tail and establishes silencing 3. deadenylation occurs, eIF4G unbinds from PABPC and mRNA loop, and it opens up 3a. add decapping complex which leads to mRNA decay via Xrn1 (5'->3') OR 3b. add deadenylase complex, which leads to translocational repression and translational inhibition with short poly A tail
How do the Pfizer and Moderna vaccines work?
1. mRNA sequence that codes for spike protein is generated 2. RNA is put into lipid coating for delivery 3. cells read info in mRNA to make spike protein themselves 4. protein spurs immune system to make antibodies against real virus when it arrives
What three mechanisms prevent mistakes (i.e., mutations) during DNA replication?
1. nucleotide selectivity (DNAP add the wrong nucleotide 1/10000) 2. proofreading (DNAP remove wrong nucleotides, reducing error rate to 1/1000000) 3. mismatch repair (wrong nucleotides are corrected, reducing error rate to 1/10000000000)
4 terms for amino acids linked by peptide bonds?
1. protein (> 100 aminos) = large 2. polypeptide (50-100 aminos) = small 3. peptide (20-50 aminos) = small 4. oligopeptide (2-20 aminos) = small
steps for translation initiation in bacteria
1. selection of initiation codon by base pairing between 16S rRNA and mRNA 2. recruitment of ternary complex to small ribosomal subunit P site 3. assembly of large ribosomal subunit after GTP hydrolysis
how does HIV replicate itself?
1. virus has 2 copies of RNA genome, enters cells and releases genome into cytoplasm, replication carried out by reverse transcriptase 2. RT -> 1x DNA -> 2x DNA, goes into nucleus and gets integrated into genome 3. then transcribed into viral mRNA and RNA genome 4. then translated into viral polyproteins that are cleaved, leaves cell to produce another virus
what are the 4 ways that proteins are detected?
1. western blot analysis...detects/quantifies in vitro via fractionating, transferring, probing, and detection 2. immunofluorescence...detects in vitro via DAPI stains, antibodies detect TAF1/TAF9, localize to different places 3. epitope tags...detect overexpressed proteins 4. mass spectrometry...identifies and quantifies proteins
what determines the site of termination in bacteria?
10 ter sites (terminus). they're bound by Tus protein. DNAP 3 can pass ter sites that are oriented in same direction as its movement, but are stalled by ter sites in opposite orientation...makes it so that 1 fork can arrive before the other and wait for the 2nd fork, or forks can arrive at same time
what's true of the rate of translation?
17-21 amino acids per second in bacteria 6-9 amino acids per second in eukaryotes
How many different codons make Phe, Tyr, His, Gln, Asn, Lys, Asp, Glu, and Cys?
2
What would a test tube of 2nd generation DNA grown in light nitrogen look like after 3 generations?
2nd generation ratio of light:intermediate = 2:2 blue/yellow yellow yellow blue/yellow 3rd generation ratio of light:intermediate = 6:2 blue/yellow yellow yellow yellow yellow yellow yellow blue/yellow
How many different codons make Ile?
3
Translation in bacteria involves all of the following types of RNA EXCEPT:
5S rRNAs tRNAs mRNAs 23S rRNA *5.8S rRNA*
How many codons make Leu, Ser, and Arg?
6
how many amino acid codons are there?
61. 3 stop codons (UGA, UAG, UAA) and 1 start codon (AUG)
how many codons are there?
64
Replication of the SARS-CoV-2 genome involves all of the following proteins EXCEPT:
A protein that unwinds double-stranded RNA A protein that binds single-stranded RNA A protein complex that synthesizes RNA using RNA as a template *A protein that binds the origin of replication* Proteins that increase the processivity of RNA-dependent RNA polymerase
Which of the following statements about the cell cycle in eukaryotes is FALSE?
A typical cell cycle contains G1, S, G2, and M phases Endomitosis is an atypical cell cycle where cells enter mitosis but do not divide Endocycle is an atypical cell cycle that does not contain M phase Endoreduplication produces polyploid cells *In endoreduplication, DNA replication occurs more than once per cell cycle*
List the molecular interactions that occur during translation initiation in bacteria
A. RNA-RNA base pairing fMet-tRNAfMet anticodon base pairing to mRNA AUG codon mRNA Shine-Dalgarno base pairing to 16S rRNA B. Protein binding to RNA IF2 binding to fMet-tRNAfMet Ribosomal proteins binding to rRNAs C. Protein binding to protein IF1, IF2, and IF3 binding to 30S ribosome subunit 50S ribosome subunit binding to 30S ribosome subunit
Kozak sequence
ACCAUGG
All of the following are types of non-bulky DNA damage EXCEPT:
Alkylation Oxidation Deamination Depurination *Base mismatch*
Describe TWO cellular factors that are required for SARS-CoV-2 replication. For example, cellular ribosomes are required for translation of viral proteins. SARS-CoV-2 does not encode its own ribosomes and thus relies on the 80S cellular ribosome for translation.
All of the cellular eIFs, eEFs, and RFs are required for translation of viral proteins. SARS-CoV-2 does not encode its own translation factors. Cellular poly(A) polymerase is required for stability of viral RNAs and translation of viral proteins. SARS-CoV-2 does not encode its own poly(A) polymerase. Cell surface receptors such as ACE2 are require for SARS-CoV-2 to enter cells and replicate. Cellular nucleotides, amino acids, and tRNAs are required for the synthesis of viral RNAs and proteins.
Hypothesize whether all of the origins of replication in a eukaryotic chromosome would need to be used to fully replicate the chromosome.
All of the origins would not be needed. As shown in bacteria, a single origin that starts bidirectional replication is sufficient to fully replicate a chromosome. Nevertheless, a minimum number of origins and the space of the origins are required to fully replicate the chromosome in the amount of time allocated to S phase of the cell cycle.
Hypothesize whether all of the origins of replication in a eukaryotic chromosome would need to fire at the same time to fully replicate the chromosome.
All of the origins would not need to fire at the same time. In theory, origins could fire at different times, with early firing origins replicating more of the genome than late firing origins.
Which of the following mutations would generate a frameshift?
An insertion of 10 base pairs in a 3' UTR An insertion of 6 base pairs in an ORF A deletion of 3 base pairs in an ORF *An insertion of 2 base pairs in an ORF* A substitution of one base for another base pair in an ORF
How does translation of AUG codons differ between initiation and elongation?
BACTERIA Initiation: tRNA = fMet-tRNA^fMet ternary complex = IF2-GTP tRNA delivery site = P site AUG IDd by = base pairing between mRNA Shine-Delgarno sequence and 16S rRNA Elongation: tRNA = Met-tRNA^Met ternary complex = EF-Tu-GTP tRNA delivery site = A site AUG IDd by = continuous translation EUKARYOTES Initiation: tRNA = Met-tRNAi^Met ternary complex = eIF2-GTP tRNA delivery site = P site AUG IDd by = ribosomal scanning of 5' UTR Elongation: tRNA = Met-tRNA^Met ternary complex = eIF1A-GTP tRNA delivery site = A site AUG IDd by = continuous translation
Which of the following statements about ribosomes is FALSE?
Bacterial 30S and eukaryotic 40S subunits serve similar functions Bacterial 50S and eukaryotic 60S subunits serve similar functions Ribosomes are ribonucleoprotein particles (RNPs) *The large and small ribosomal subunits are always complexed together* Ribosomes can associate with the endoplasmic reticulum in eukaryotes
Why do bacteria not need telomerase for DNA replication?
Bacterial chromosomes are circular, so there are no ends that would need to be replicated by telomerase
Which of the following events DOES NOT occur during translation initiation?
Base pairing between the mRNA Shine-Dalgarno sequence and 16S rRNA, in bacteria Base pairing between the initiator tRNA and an AUG codon in the ribosome P site *Assembly of the large ribosome subunit prior to assembly of the initiator tRNA* Delivery of the initiator tRNA to the ribosome as part of a ternary complex Ribosome scanning of the mRNA for an AUG codon, in eukaryotes
All of the following events occur during translation in eukaryotes EXCEPT:
Binding of an eIF to the mRNA 5' m7G cap *Delivery of aminoacyl-tRNAs to the ribosome A-site during initiation and elongation* Circularization of the mRNA Scanning of the 5' UTR for an AUG codon Recognition of all three stop codons by a single RF
A nonsense mutation generating a UGA stop codon was produced in the coding region of a gene. If the mutation was due to a base transition, which amino acids may have been encoded by the original codon? Also, what type of DNA damage may have led to the mutation?
CGA (Arg) to UGA (stop) and UGG (Trp) to UGA (stop). Both mutations could be caused by deamination of cytosine.
Why is it unlikely that continuous replication of both DNA strands occurs but is yet to be discovered?
Continuous replication is unlikely to occur because it would require DNA and RNA polymerases that synthesize in the 3'-to-5' direction. All of the characterized DNA and RNA polymerases from bacteria and eukaryotes synthesize in the 5'-to-3' direction. Furthermore, synthesis in the 3'-to-5' direction might run into trouble because phosphates at the 5' end of the growing chain could easily by hydrolyzed, which would block addition of new nucleotides at the 5' end because hydrolysis of phosphate bonds is required to link nucleotides.
what evidence exists to prove that the genetic code is non-overlapping and continuous?
Crick and Brener looked at the effects of shifting around codons on genotype and phenotype, found that it is
Which of the following definitions is also also INCORRECT?
DNA damage is a physical or chemical abnormality in the structure of DNA *Induced mutations are mutations caused by the cellular environment* Mutations are changes in the base pair sequence of DNA that cannot be repaired Germline mutations occur in gametes Somatic mutations occur in cells other than gametes
Which of the following mechanisms DOES NOT protect eukaryotic chromosomes from mutations?
DNA polymerase nucleotide selectivity DNA polymerase proofreading activity Extension of chromosome ends by telomerase The t-loop structure at telomeres *Incorporation of a nucleoside reverse transcriptase inhibitor (NRTI)*
Which of the following statements about DNA repair is FALSE?
DNA repair mechanisms often involve detection, incision, excision, synthesis, and ligation steps Base excision repair begins with removal of the damage base *In translesion synthesis, the damage DNA is excised* In nonhomologous end joining, Ku proteins detect the double-strand break in DNA Nucleotide excision repair has transcription-independent and transcription-dependent mechanisms
Why must mismatch repair take place during DNA replication?
DNA replication allows the repair machinery to distinguish between the wild-type template DNA strand and the mutant newly replicated DNA strand.
All of the following are major sources of DNA damage that lead to mutations EXCEPT:
DNA replication errors Cellular environment Exogenous chemical agents Exogenous physical agents *Transcription errors*
what causes spontaneous mutations?
DNA replication errors. can be mispairing during DNA replication (normal base pairs become tautomeric, ionized base pairs, wobble base pairs, transitions (purine to purine) or transversions (purine to pyrimidine)) cellular environment. H2O and reactive O in cells causes DNA damage -> mutations
Which of the following statements about DNA replication in bacteria is FALSE?
DNA replication initiates at an AT-rich region of the genome called oriC Synthesis of the leading strand is continuous DNA replication can take longer than the cell doubling time *DNA replication can initiate at multiple places in the genome* Synthesis of the lagging strand is discontinuous
Which of the following statements about DNA replication is FALSE?
DNA replication is discontinuous on the lagging strand DNA replication is continuous on the leading strand DNA ligase joins Okazaki fragments *DNA is synthesized in the 3' to 5' direction* DNA replication causes supercoiling of DNA
Hypothesize why non-dividing cells might have fewer mutations than dividing cells in eukaryotes?
DNA replication, which is a source of DNA damage through base mismatches and slippage, does not occur in non-dividing cells, so mutations cannot occur from this source. Alternatively, non-dividing cells may have more effective DNA repair mechanisms or lower rates of spontaneous DNA damage.
which special bacterial/eukaryotic DNAP are involved in DNA repair?
DNAP 1, 2, 3, 5 alpha, delta, epsilon, etc.
Which of the following protein-related processes DOES NOT take place?
Degradation of polyubiquitinated proteins by the proteasome *Phosphorylation of tryptophan* Localization of proteins to particular places in cells by signal sequences Inhibition of translation by miRNAs Modification of proteins by addition of a sugar or a lipid
What amino acids are encoded by a repeated AGA sequence?
Depending on reading frame: Arg (AGA) Glu (GAA) Lys (AAG)
All of the following are common steps in DNA repair pathways EXCEPT:
Detection Excision Synthesis Ligation *Strand invasion*
What proteins detect DNA damage in the following repair mechanisms?
Direct damage: CPD photolyase and MGMT Base excision repair: (Bacteria and eukaryotes) DNA glycosylase Nucleotide excision repair: (Bacteria) UvrA/UvrB and UvrA/UvrB/RNAP (Eukaryotes) XPE/XPC and CSA/CSB/RNA pol II Non-homologous end joining: (Bacteria and eukaryotes) Ku proteins
Which of the following DNA repair mechanisms DOES NOT take place during G1 phase of the cell cycle?
Direct repair Base excision repair Nonhomologous end joining Nucleotide excision repair *Mismatch repair*
Describe TWO factors that may be targets of drugs to prevent SARS-CoV-2 replication. For example, drug that inhibit the activity of viral protease enzymes would block viral replication by preventing generation of the RNA-dependent RNA polymerase (RdRP) that is essential for replication of the viral genome.
Drugs that inhibit the activity of viral RdRP would block viral replication by preventing replication of the viral genome. The drugs may be ribonucleotide or ribonucleoside analogs that cause chain termination similar to NRTIs that are used to block replication of the HIV genome. Drugs that inhibit the activity of viral RNA capping enzymes may block viral replication by preventing translation of the viral genome and viral mRNAs. Without a cap, the cellular cap binding protein eIF4E could not bind the RNAs and promote circularization of the mRNA and scanning for the start codon AUG. Drugs that inhibit viral helicase may block viral replication by preventing replication of the viral genome. Helicase activity is critical for unwinding of double-stranded RNA during viral genome replication. Drugs that inhibit viral processivity factor may block viral replication by preventing replication of the viral genome. Processivity factor activity is critical for preventing RdRP from dissociating from the genomic RNA before it completely replicates the genome.
Which of the following statements about translation elongation or termination is FALSE?
During elongation, tRNAs are delivered to the ribosome A site Release factors bind stop codons Peptide bond formation occurs in the large ribosomal subunit *Ribosomal proteins carry out peptide bond formation* GTPases play roles during elongation and termination
purposes of the 3 binding sites for tRNA in the small ribosomal subunit?
E site = exit P site = peptidyl site A site = aminoacyl-tRNA binding site
Why might an unphosphorylated IKBa not be ubiquitinated?
E3 ligase only binds phosphorylated IKBa, and in many examples it isn't.
eukaryotic cell cycle
G1 = cell growth S = DNA replication G2 = cell growth M = cell division
who were the solvers of the genetic code puzzle, and learned to match codons and amino acids?
Holley (isolated and mapped structure of tRNAs) Nirenberg (synthesized UUU RNA and found that it only made 1 protein, Phe) Khorana (chemically synthesized RNA with specific sequences, and found the rest of the genetic code that way)
Describe the mechanism that limits DNA replication to once per cell cycle in eukaryotes?
In eukaryotes, prereplication complexes (pre-RCs) contain ORC (Origin Recognition complex), replication helicase (MCM2-7), Cdc6 and Cdt1, which are involved in assembly of MCM2-7 onto DNA at origins. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period because Cdc6 and Cdt1 are only present at this time. They get degraded by the proteasome early in S phase and Cdc6 is not reexpressed until G1 phase.
Why do intercalating agents such as acridine orange commonly produce amorphic mutations?
Intercalating agents cause double-strand DNA breaks that can be repaired by nonhomologous end joining (NHEJ), which inserts and deletes base pairs at the site of the break. If the break is in the coding region of protein-coding genes, the insertion or deletion will likely lead to a translation frameshift, which changes the sequence of the protein downstream of the mutation and will probably lead to a premature translation termination codon. Thus, the encoded protein is likely to be nonfunction and the mutation would be amorphic (i.e., a complete loss-of-function).
Which of the following statements about DNA replication is also FALSE?
It is bidirectional It requires RNA primers for both leading and lagging strand synthesis It begins at origins *It occurs more than once per cell cycle in polyploid cells* It requires multiple DNA polymerases
Which of the following statements about the Ames test is TRUE?
It is carried out with wild-type bacteria It is used to identify sources of spontaneous mutations It is used detect base substitutions, not insertions and deletions It does not require bacterial growth *It is used to identify compounds that induce mutations*
All of the following are properties of the genetic code EXCEPT:
It is degenerate (i.e., an amino acid can be coded for by more than one codon) It is universal (i.e., all organisms use the same genetic code) There are 64 codons *It determines what amino acid will be attached to what tRNA* It contains one codon for methionine
Which of the following statements about telomerase is TRUE?
It is present in both bacteria and eukaryotes *It prevents the progressive shortening of chromosomes with each round of DNA replication* It uses DNA as a template and synthesizes DNA Its expression remains constant throughout the life of a somatic cell It uses the 5' end of a DNA strand as a primer
Which of the following statements about peptide bond formation is FALSE?
It occurs within the large ribosomal subunit *It transfers the polypeptide to the ribosome P site* It is catalyzed by rRNA It involves positioning of tRNAs by base pairing to rRNA It produces water as a byproduct
What anticodon sequence base pairs to the GUU codon in E. coli?
It's a wobble base pair with CAG (3 to 5) for tRNA, and GUU (5 to 3) for mRNA
All of the following enzymes are writers of post-translational modifications EXCEPT:
Kinases Methyltransferases Acetyltransferases *Phosphatases* Glycosylases
Complete the table regarding leading and lagging strands.
Leading strand = continuous, synthesis in 5' to 3' direction Lagging strand = discontinuous, synthesis in 5' to 3' direction
Predict what happens to the number of mutations as people age (line A, B, or C in the graph below), and provide a rationale for your answer.
Line A is correct. The number of mutations increases with age. The graph below shows the frequency of a specific mutation in the FGF3 gene in sperm DNA with age. The graph would be similar for mutations in somatic cells such as muscle cells. The reason that the number of mutations increases with age is that once mutations occur they cannot be removed, and organisms are exposed to mutagens throughout their life, so the number of mutations is always increasing.
evidence for spontaneous/induced mutations?
Luria and Delbruck in 1943: bacteria become heritably resistant to phage infection/killing. used experimental system to test if spontaneous/induced...if growing cells differently doesn't affect mutation rate, conclude that what occurred AFTER cell growth (infection) induced mutations. spontaneous = high variability induced = low variability
DNA replication occurs during which phase of the eukaryotic cell cycle?
M G1 G2 DNA replication takes place in all of these cell cycle phases *S*
How would you test if the bold sequence below is a nuclear localization signal?
MCDVLYS*KKRKRR*GGLNKEEPMSSHGDDEE A loss-of-function experiment: Delete the bold sequence and see if the protein still localizes to the nucleus. This experiment tests the necessity of the sequence to localize the protein to the nucleus. Another loss-of-function experiment: Mutate the positively charged bolded amino acids to non-charged amino acids such as alanine or negatively charged amino acids such as aspartate and see if the protein still localizes to the nucleus. This experiment tests the necessity of the sequence to localize the protein to the nucleus. A gain-of-function experiment: Put the bold sequence into a protein that normally localizes to the cytoplasm and see if it now localizes to the nucleus. This experiment tests the sufficiency of the sequence to localize proteins to the nucleus.
experimental evidence that DNA replication is semiconservative?
Messelson-Stahl experiment...saw 1/2 DNA with N15, and other 1/2 with N14
Which of the following statements about mutations is FALSE?
Mutations can be spontaneous or induced The number of mutations increases with age The Ames test identifies compound that cause mutations *Mutation and DNA damage mean the same thing* DNA replication errors can lead to mutations
Which of the following statements about DNA repair is also FALSE?
Mutations in genes encoding DNA repair proteins cause diseases in humans Some repair pathways in eukaryotes only take place during S phase of the cell cycle *All DNA repair mechanisms involve removal of the damaged nucleotides* Transcription can detect DNA damage to activate nucleotide excision repair in bacteria and eukaryotes Mismatch repair requires a mechanism that distinguishes the template and newly synthesized DNA strands
Why might NRTIs inhibit HIV reverse transcriptase but not cellular DNA polymerases?
NRTIs were designed to have higher affinity for HIV reverse transcriptase than cellular DNA polymerases involved in DNA replication and repair. The idea of DNA polymerases having different affinities for different nucleotides is related to nucleotide selectivity during DNA replication. DNA polymerases have a binding pocket for nucleotides and DNA base pairs, and nucleotides and base pairs need to fit into the pocket in a particular way to be used efficiently.
Which amino acids are most likely to be modified and why?
Non-polar amino acids are less likely to be modified because they are often located in the hydrophobic core of proteins, away from the aqueous environment. In contrast, polar amino acids are more likely to be modified because they are often located on the surface of protein, exposed to the aqueous environment. Slide 15 of the lecture shows that modifications of polar amino acids such as serine, threonine, tyrosine, lysine, and asparagine are most common
What probes are used to detect DNA, RNA, and protein in vivo and in vitro?
Nucleic acids are detected in vivo and in vitro by hybridization with complementary nucleic acids and proteins are detected in vivo and in vitro using antibodies that bind specific proteins. DNA, in vivo = fluorescence in situ hybridization (FISH), probe is DNA/RNA RNA, in vivo = in situ hybridization, probe is DNA/RNA protein, in vivo = immunofluorescence, probe is antibody DNA, in vitro = southern blot, probe is DNA/RNA *OR* polymerase chain reaction (PCR), probe is DNA primer RNA in vitro = northern blot, probe is DNA/RNA *OR* reverse transcription PCR (RT-PCR), probe is DNA primer protein, in vitro = western blot, probe is antibody
Which of the following mechanisms DOES NOT function to reduce the rate of DNA replication errors?
Nucleotide selectivity of DNA polymerases Proofreading activity of DNA polymerases *Nucleotide excision repair* Mismatch repair
what's translated from the viral genome of COVID-19?
ORF1a and ORF1b. +gRNA acts as mRNA for their translation
4Which of the following statements about protein ubiquitination is FALSE?
Polyubiquitination promotes protein degradation by the proteasome *Polyubiquitin chains only link ubiquitin molecules through lysine 11* Ubiquitin is added to lysine in proteins Ubiquitination involves E1, E2, and E3 enzymes Ubiquitin is linked to proteins through its C-terminal glycine
Which of the following statements about protein modification is FALSE?
Protein modifications can alter the cellular localization of proteins *Most protein modifications occur on the inside of proteins* Protein modifications can alter protein-protein interactions Most protein modifications are reversible Protein modification can alter the biochemical function of proteins
Production of SARS-CoV-2 proteins requires all of the following events EXCEPT:
Proteolysis of polyproteins Programmed translation frameshift *Base pairing between rRNA and translation regulatory sequences* Translation of subgenomic RNAs Translation of genomic RNA
How would the base excision repair mechanism differ between repair of damage due to base alkylation versus depurination?
Repair of depurination would not require the first step in base excision repair (i.e., removal of the base by glycosidic bond cleavage by DNA glycosylase) because the base is already removed.
Which of the following molecular processes IS NOT used by SARS-CoV-2 for its replication?
Replication of new genomes using old genomes as templates Translation of mRNAs Post-translational cleavage of proteins Transcription of mRNAs using the genome as a template *Splicing of pre-mRNAs*
SARS-CoV-2 replication requires all of the following cellular molecules EXCEPT:
Ribosomes *RNA polymerases* Translation initiation factors Nucleotides Amino acids
when does replication occur in the cell cycle of eukaryotes? how does it only occur 1x per cell cycle?
S phase. and there are 2 key protein factors for replication only present at the end of G1: Cdc6, and Cdt1...both get ubiquitinated and degraded by proteasome, preventing the assembly of new replisomes
How is the synthesis of SARS-CoV-2 and human proteins different?
SARS-CoV-2 proteins are translated using the genome as a template. A translation frameshift is required for the synthesis of some SARS-CoV-2 proteins Some SARS-CoV-2 proteins are synthesized as polyproteins that are cleaved into individual proteins by proteases.
What are similarities and differences between base excision repair (BER) and mismatch repair (MR)?
Similarities: Both BER and MR involve detection, excision, incision, synthesis, and ligation steps In MR and long patch BER a stretch of nucleotides is removed to repair the damage. Differences: They repair different types of DNA damage: BER repairs damage to or loss of a base, while MR repairs a base mismatch as well as insertion and deletion loops that result from DNA polymerase slippage during replication of repetitive DNA sequences BER occurs during G1 phase, and MR occurs during S phase BER and MR involve different proteins In short patch BER, only a single nucleotide is excised
What are the similarities and differences between nonhomologous end joining (NHEJ) and homologous recombination?
Similarities: Both NHEJ and HR repair double-strand breaks in DNA Both NHEJ and HR involve detection, excision, and ligation steps Differences: HR leads to accurate repair, while NHEJ is error-prone leading to mutations HR involves a synthesis step that uses undamaged homologous DNA as a template, while NHEJ does not involve a synthesis step They involve different proteins NHEJ takes place during G1 phase and HR takes places during S and G2 phases
What are similarities and differences between RFs and tRNAs?
Similarities: Overall shape Bind in the ribosome A-site Span from A-site to the peptidyl-transferase center Differences: tRNAs are RNAs, RFs are proteins tRNAs bind codons through base pairing, RFs bind codons through protein-RNA interactions tRNAs carry an amino acid, RFs promote cleavage of the protein from the tRNA in the P-site
List THREE similarities and THREE difference between translation in bacteria and eukaryotes.
Similarities: Use the same genetic code Use the same 20 amino acids Continuous and nonoverlapping Almost always starts at an AUG codon The AUG codon is bound by the small ribosomal subunit before the large ribosomal subunit joins Use analogously functioning IFs, EFs, and RFs GTP hydrolysis by GTPases cause conformation changes that promote the next step in translation Initiated by delivery of a charged tRNA to the ribosome P site Involve ribosomes that contain several rRNAs and many proteins Aminoacyl-tRNA synthetases pair tRNAs with amino acids Base pairing between tRNAs anticodons and mRNA codons can involve Watson-Crick and wobble base pairs at the third position of the codon A-to-I editing can increase the wobble base pairing possibilities (but this occurs to a greater extent in eukaryotes than in bacteria) Charged tRNAs are delivered to ribosomes in a ternary complex Ribosomes are recycled after termination by recycling factors Differences: Transcription and translation occur at the same time in bacteria but at different times in eukaryotes The AUG start codon in bacteria is identified by base pairing between the mRNA Shine-Dalgarno sequence and 16S rRNA but in eukaryotes it is identified by scanning of the small ribosomal subunit In bacteria, two RFs recognize the three stop codons but in eukaryotes one RF recognizes all three stop codons In eukaryotes, translation involves binding of factors to the 5' and 3' ends of the mRNA (i.e., cap binding protein (eIF4E) and poly(A) binding protein (PABP), respectively) and circularization of the mRNA, but this does not occur in bacteria In bacteria the initiator tRNA is tRNAfMet but in eukaryotes it is tRNAiMet, and the initiating amino acid in bacteria is fMet but in eukaryotes it is Met
Given what you know about the structure and function of telomerase, provide a plausible model to explain how a species could exist with a combination of two different repeats (for example TTGGGG and TTGTGG) on each of their telomere.
Since the telomerase RNA determines the sequence of telomeres, there are either two telomerase RNAs (one for each repeat sequence) or both sequences occur in a single telomerase RNA.
Bacterial ribosomes contain all of the following EXCEPT:
Small and large subunits RNAs and proteins *A protein enzyme that forms peptide bonds* E, P, and A binding sites for tRNAs 16S rRNA sequences that base pair with mRNA Shine-Delgarno sequence
Which of the following statements about DNA polymerases is FALSE?
Some DNA polymerases function in both DNA repair and DNA replication *Bacteria have a DNA polymerase that is dedicated to replication of chromosome ends* DNA polymerases differ in their processivity Some DNA polymerases have 3' to 5' exonuclease activity for proofreading Some DNA polymerases in eukaryotes function in specific DNA repair pathways
translesion synthesis bypass at stalled DNA replication forks
TLS polymerases are different from replication polymerases: 1. don't stall at damaged bases because have large active sites that can accomodate them 2. can be error prone because lack 3' to 5' proofreading activity 3. low processivity; can only add a few nucleotides before f*cking off of DNA template steps: replicative polymerase stalls at lesion and detects, TLS polymerase replaces prior and synthesizes, prior re-joins and continues synthesis
Describe TWO functions of the CCA sequence at 3' end of all tRNAs.
The 3' OH of the A in the CCA serves as the attachment site for an amino acid The CCA base pairs to rRNA to position tRNAs in the ribosome P and A sites for peptide bond formation The CCA is bound by elongation factors that deliver charged tRNAs to the ribosome A site
How do genome replication of SARS-CoV-2 and a human cell differ?
The SARS-CoV-2 genome is ssRNA and the human genome is dSDNA, so the mechanism of genome replication must differ. Replication of the SARS-CoV-2 genome takes place in the cytoplasm, whereas replication of the human genome takes place in the nucleus. SARS-CoV-2 relies on cellular processes of the host cell for its genome replication, whereas human cells replicate their genome autonomously.
Why might the size of the nuclei of four tomato cells differ?
The cells get increasingly larger, and thus have different amounts of DNA. See graph below, which shows that the size of nuclei increases as the ploidy increases. This makes sense because bigger nuclei are needed to hold more DNA. 2N, 4N, 8N, 16N
Which of the following statements about the genetic code is FALSE?
The genetic code is universal There are 61 amino acid codons and 3 stop codons The genetic code is degenerate *Bacteria and eukaryotes have different genetic codes* The genetic code is tolerant to mutations
Describe TWO reasons why the amount of an mRNA in a eukaryotic cell does not always represent the amount of the encoded protein.
The mRNA might not be efficiently transported to the cytoplasm and thus cannot be translated The protein may be synthesized but rapidly degraded through the ubiquitin-proteasome mechanism Translation initiation may be inefficient because of an AUG start codon that is in a poor context (i.e., Kozak sequence) or there is an RNA structure in the 5' UTR that inhibits ribosome scanning and cannot be effectively melted by the eIF4A helicase There may be signaling pathways that lead to post-translational modification of translation regulatory factors or the ribosome that upregulate or downregulate translation of the mRNA
Describe why the mutation rate in human cells might increase with age?
The mutation rate might increase with age because there is either an increase in the rate of DNA damage or a decrease in the efficiency of DNA repair.
Based on the sources of DNA damage, do you think that the rate of DNA damage is the same or different in different cell types of a multicellular organism? Provide an explanation to justify your answer.
The rate of DNA damage will differ among different cell types because cells have different amounts of endogenous molecules that cause DNA damage (e.g., different amounts of reactive oxygen species produced by mitochondria) and they are exposed to different amounts of chemical and physical agents that cause DNA damage (e.g., lung cells are exposed to chemicals in cigarette smoke more than muscle cells, and skin cells are exposed to ionizing radiation from the sun more than muscle cells).
When the genetic code was being determined, it was found that (GUC)n encodes three homopeptides but (GUA)n only encodes two homopeptides, explain why.
The three reading frames for GUC are GUC GUC GUC GUC, UCG UCG UCG UCG, and CGU CGU CGU CGU, which encode poly-valine, poly-serine, and poly-arginine, respectively. The three reading frames for GUA are GUA GUA GUA GUA, UAG UAG UAG UAG, and AGU AGU AGU AGU, which encode poly-valine, nothing (all stop codons), and poly-serine. Thus, only two of the reading frames encode proteins.
What happens if a tRNA is charged with the wrong amino acid?
The wrong amino acid would get incorporated into a protein. Additionally, depending on the location of the amino acid in the protein and the structure of the amino acid relative to correct amino acid, the wrong amino acid could affect the cellular localization of the protein, the enzymatic activity of the protein, the post-translational modifications of the protein, the interactions of the protein with other molecules (DNA, RNAs, and proteins), or the stability of the protein.
Why might DNA viruses have high rates of DNA replication errors?
There are three possible answers: (1) Viral DNA polymerases may have low nucleotide selectivity, so they incorporate the wrong nucleotide at a high rate during DNA replication (2) Viral DNA polymerases may lack proofreading activity, so they cannot remove incorrectly added nucleotides during DNA replication (3) Incorrectly added nucleotides may not be able to be corrected by the cellular mismatch repair mechanism. All of these mechanisms contribute to the high mutation rate of viruses
Why isn't a C-to-U transition that is caused by deamination and creates a base mismatch (i.e., conversion of a C-G base pair to a U-G mismatch) repaired by mismatch repair?
There are two possible reasons. First, mismatch repair might not detect the damage because it only operates during DNA replication (i.e., S phase of the cell cycle). Second, the MutS ortholog in eukaryotes might not recognize the mismatch because dUMP is not incorporated into DNA during DNA replication.
Which of the following IS NOT a property of nucleoside reverse transcriptase inhibitors (NRTIs)?
They are triphosphorylated by cellular enzymes They are preferentially used as substrates by HIV reverse transcriptase relative to cellular DNA polymerases *They contain a 2' OH* They lack a 3' OH They cause chain termination during replication of the HIV genome
All of the following are properties of aminoacyl-tRNA synthetases EXCEPT:
They attach amino acids to tRNAs *There is one for each tRNA* They require ATP for their activity They discriminate among amino acids based on their size and shape They discriminate among tRNAs by interactions with the acceptor stem and anticodon
Which of the statements about signaling pathways is FALSE?
They control the transcription of specific genes They control the translation of specific mRNAs They transfer information between proteins by post-translational modifications They transfer information by altering interactions between proteins *They only occur in multicellular organisms*
Describe TWO properties of the Spike mRNA that Pfizer and Moderna needed to consider when designing their vaccines? For example, they needed to include an open reading frame that encoded the dominant form of the Spike protein that was present at that time. With the emergence of variants such as omicron, the open reading frame sequence may need to be changed to maintain the effectiveness of the vaccine.
They needed to include an m7G cap at the 5' end of the mRNA to prevent mRNA decay and promote translation. They needed to consider the sequence at -4 and +3 relative to the translation start site AUG to best match the Kozak consensus sequence and optimize translation initiation of the mRNA. They needed to consider the sequence of the 3' UTR to eliminate binding sites for factors such as miRNAs that would block translation and promote decay of the mRNA. They need to include a poly(A) tail at the 3' end of the mRNA to prevent mRNA decay and promote translation. They needed to include sequences encoding a signal peptide at the N-terminus of the Spike protein so that it correctly localized to the plasma membrane of cells.
Which of the following statements about miRNAs is FALSE?
They promote mRNA decay and block mRNA translation *They are encoded by bacteria* They are bound by proteins They base pair to mRNA 3' UTRs They are encoded by eukaryotic organisms
Translesion synthesis DNA polymerases are similar to replication DNA polymerases in all of the following ways EXCEPT:
They require a primer to begin DNA synthesis They synthesize DNA in the 5' to 3' direction They are found in both bacteria and eukaryotes They interact with beta-clamp *They stall at damaged bases*
Which of the following statements about GTPases in translation is FALSE?
They stimulate release of the completed polypeptide They control assembly of the large ribosomal subunit *They promote unwinding of double-stranded regions of mRNA* They ensure rapid and accurate incorporation of amino acids into the growing polypeptide They promote translocation of the ribosome
How would you determine if telomerase activity is necessary for cancer to persist or sufficient to cause cancer?
To determine necessity, you could perform a loss-of-function experiment that involved blocking expression of telomerase, either the telomerase protein or RNA. If the cells remained cancerous, you could conclude that telomerase is not necessary to maintain the cancer phenotype. Alternatively, if the cells were no longer cancerous, you could conclude that telomerase is necessary to maintain the cancer phenotype. To determine sufficiency, you could perform a gain-of-function experiment that involved misexpressing telomerase (both the telomerase protein and RNA) in mitotically active somatic cells that are not cancerous. If the cells do not become cancerous, you could conclude that telomerase is not sufficient to cause cancer. Alternatively, if the cells do become cancerous, you could conclude that telomerase is sufficient to cause cancer.
Which of the following definitions is INCORRECT?
Transitions change a purine to a purine or a pyrimidine to a pyrimidine Transversions change a pyrimidine to a purine or a purine to a pyrimidine Nonsense mutations change an amino acid codon to a stop codon *Conservative mutations do not change the amino acid specified by a codon* Frameshift mutations change the codons downstream of the mutation
Describe what would happen in cells if eIF2-GDP cannot be converted to eIF2-GTP.
Translation of most if not all mRNAs would be blocked because eIF2-GTP is part of the ternary complex that contains Met-tRNAMet and initiates translation at an AUG codon.
How might viral miRNAs interfere with the ability of host cells block viral replication?
Viral miRNAs might block the translation of host mRNAs that promote the inflammatory response that inhibits viral replication
Which of the following statement about codon-anticodon wobble base pairing is TRUE?
Wobble base pairing can occur at the first position within a codon Wobble base pairing can occur at the second position within a codon A and G can form a wobble base pair A and U can form a wobble base pair *Wobble base pairing can occur at the third position within a codon*
what does GTP hydrolysis promote?
assembly of the 60S subunit and release of eIFs. with 60S subunit added + eIF5B-GTP, lose eIF1 + eIF1A + eIF3 + eIF5 and eIF2-GDP + Pi and eIF5B-GDP + Pi
where does A to I editing occur in eukaryotes?
at all tRNA anticodons with the sequence 5'-ANN-3'
aminoacyl-tRNA synthetase
attaches amino acids to tRNAs. there are ~20 of these, 1 for each amino acid. can act on multiple tRNAs and have high fidelity/accuracy. the tRNA meets up with it, and the amino gets created on the other side. step 1 = amino acid gets activated and loses 2 phosphates. step 2 = AMP created. can act in pool of amino acids, or pool of tRNAs. error rate = 1/10,000
mismatch repair of DNA replication errors
bacteria: MutS + MutL+MutH do incision, strand is separated and excised by UvrD and exonuclease, DNAP 3 synthesizes, and DNA ligase ligates eukaryoes: similar, except strand specificity of endonuclease is directed by PCNA, not DNA methylation
nucleotide excision repair of pyrimidine dimers and bulky adducts
bacteria: UvrB acts as helicase to separate strands once detected, UvrC cleaves 8 nt 5' and 4-5 nt 3' of damage for incision, UvrD removes 12-13 nt region for excision, then DNAP 1 acts for synthesis and DNA ligase acts for ligation eukaryotes: XPB/XPD of TFIIH act as helicases to separate strands once detected, XPF-ERCC1 cleaves 5' of damage and XPG of TFIIH cleaves 3' of damage for incision, XPG removes ~27 nt region for excision, RPC-clamp loader and PCNA-B clamp act alongside DNAP delta/epsilon during synthesis, and Lig3/XRCC1 ligate
homologous recombination repair of DNA 2x strand breaks
bacteria: detection and excision by RecA or Rad51, DNAP synthesizes and DNA ligase ligates eukaryotes: exonucleases do excision, recombinases come in to synthesize D loop, then splits into 2 pathways: 1. 2x strand break repair (DSBR) = 2nd end capture, DNA synthesis and ligation, then holiday junction resolution, and more synthesis and ligation to get a very mixed end result of recombination 2. synthesis-dependent strand annealing (SDSA) = strand displacement and annealing, then DNA synthesis and ligation to get a some small recombination *not error prone; doesn't introduce errors into DNA*
base excision repair of non-bulky DNA damage
bacteria: detection via DNA glycosylase, incision via AP endonuclease, excision via DNA phosphodiesterase, synthesis via DNAP 1, ligation via DNA ligase eukaryotes: detection via DNA glycosylase, incision via AP endonuclease, splits into 2 for short patch (synthesis insertion and excision via flap endonuclease and DNA ligase) or long patch (excision and synthesis via pol B and ligation via DNA ligase)
how is bacterial translation of mRNAs different than that of eukaryotes?
bacterial mRNA is transcribed from single genes, and operons. eukaryotic mRNA is only transcribed from single genes
when is mismatch repair (S) used?
base mismatch, loop
why do proteins localize to specific places in cells?
because they contain localization sequences. proteins are made in cytosol, then get moved to other places in cells. transported through nuclear pores, across membranes, and then via vesicles
oxidative damage and depurination
block base pairing, create abasic site (AP). 2000-10000 every day in human cells. pyrimidines lost less often than purines
what do NRTIs do?
block reverse transcriptase so it can't replicate viral RNA genome for HIV. based on mechanism of action of acyclovir, discovered by Elion. do so via chain termination: lack a 3' OH, so there are nucleoside (no P) analogs on deoxyribose sugar which when incorporated into replicated viral DNA by RT *ends* chain elongation. this activity requires phosphorylation by cellular enzymes in the cell cytoplasm: AZT -(TK)> AZT-MP -(dtMP-K)> AZT-DP -(NDP-K)> AZT-TP
what's true of tRNA and synthetases?
both the tRNA acceptor stem (CCA) and the anticodon loop have docking sites on the aminoacyl-tRNA synthetase
Cdt1
bound to 2nd helicase
what's true of COVID-19's replisome?
it has an error rate of 1/10000000
types of DNA mutations?
can be spontaneous (caused by DNA replication errors and damage from cellular environment) or induced (caused by DNA damage from external environmental agents) can be germline mutations (occur in gametes and are passed on to offspring) or somatic mutations (occur in all other cell types and are not passed on to offspring)
consequences of mutation?
can either be silent, missense (conservative or nonconservative), or nonsense
what can tRNAs do?
carry specific amino acids and base pair to mRNA codons. they're usually 70-100 nt long (small-ish) with their amino acid attachment site at the top, and an anticodon site (that pairs with the mRNA) at the bottom
deamination
causes base transitions: C -> U becomes U + A A -> hypoxanthine becomes H + C G -> xanthine becomes X + C 5-M cytosine -> T becomes T + A ex: C -> U occurs 100-500x a day in humans...50% of known pathogenic SNPs
senescence
cell division stops, contributes to aging and disease
endomitosis
cells enter mitosis but don't complete it or divide
what can induce mutations?
chemical and physical agents. the chemical modification of bases leads to DNA damage via alkylating agents and bulky adducts. base analogs and intercalating agents can also cause DNA damage without modifying bases. exogenous physical agents (UV light, ionizing radiation) induce pyrimidine transitions and block replication and transcription, or prevent strand break repair and create oxidative damage
what is DNA damage and what can it lead to?
chemical changes to DNA in its physical structure that CAN be repaired by cell mechanisms...can lead to mutations, which are changes in the base sequence of both DNA strands that CANNOT be repaired and get passed on to offspring
PCR test for COVID-19
collect sample, extract RNA, convert to DNA, synthesize 2nd DNA strand, do real-time PCR, and quantify DNA
mismatch repair
corrects errors that occur during DNA replication
which 2 post-translational modifications aren't reversible?
deamidation and proteolysis
what does proofreading of DNAPs do?
decrease the rate of DNA replication errors. DNAP discriminates between correct and incorrect nucleotides, has nucleotide selectivity/proofreading/mismatch repair. improves fidelity of alpha, delta, and epsilon DNAP 10-1000 fold humans: ~10-100 mutations introduced with every genome replication
RNaseH
degrades DNA strand of RNA-DNA hybrid (HIV-1 RT)
which repair mechanisms are independent of DNA replication?
direct repair of DNA damage, base excision repair of non-bulky DNA damage, nucleotide excision repair of pyrimidine dimers and bulky adducts, nonhomologous end joining repair of DNA 2x strand breaks
nonhomologous end joining (G1) and homologous recombination (S, G2)
double-strand break
how is elongation different from initiation?
during elongation, tRNA goes into P site. then 2nd amino acid is brought into A site by EF-Tu-GTP (eEF-1A in eukaryotes). GTP hydrolysis occurs and EF-Tu-GDP f*cks off, there's peptide bond formation, and then translocation moves P/A to E/P via EF-G-GTP (eEF2 in eukaryotes). Pi is lost. the ternary complex binds to the A site, and the tRNA in the E site leaves with EF-G-GDP. the cycle is repeated over and over
what's true of EF-Tu and EF-G regarding elongation?
during it, they perform repetitive functions
direct repair of DNA damage
for pyrimidine dimer, involves detection via photoreactivation (bacteria) for alkylation, involves DNA methyltransferase
what do base insertions/deletions in open reading frames cause?
frameshifts. often lead to 1 or 2 premature stop codons. however insertions/deletions in multiples of 3 don't cause frameshifts ex: deletion in CCR5 gene causes resistance to HIV infection. wild type CCR5 gene has 7 transmembrane region, mutant only has 4 so HIV can't bind to mutant CCR5 protein
neomorph
gain of function. has completely new gene activity, often in regulatory region
antimorph
gain of function. inhibits wild type gene activity. often truncates protein or alters active site
hypermorph
gain of function. more gene activity than wild type. usually missense, often in regulatory region
what does unrepaired DNA damage lead to?
genomic instability and disease...increased tendency to accumulate mutations
antigen test for COVID-19
get nasal swab sample, passes to conjugate pad with nucleocapsid protein, then to test line and control line
antibody test for COVID-19
humans produce IgM and IgG antibodies, with IgM highest in days 7-10 and IgG highest after that, but not quite as much. use lateral flow device to detect both
when do cells replicate DNA without cell division?
in rare occasions, what's known as endoreplication. still only occurs ONCE PER CELL CYCLE. creates polyploid cells ex: drosophila salivary gland cells undergo 10 rounds of DNA replication (1000N), copies all pair together (see when stain DNA with DAPI) other ex: liver cells in mice, intestinal cells of C. elegans
where does peptide bond formation happen?
in the ribosomal large subunit by rRNA
transcription of -sgRNAs
initiates at 3' end of genome, done by RdRp. proceeds until reaches TRS upstream of ORFs. it's discontinuous...-sgRNA is transferred to 5' end of genome, base pairs and binds complementary TRS. then continues to 5' end of genome to make complete -sgRNA so that +sgRNAs can be generated from it, using it as a template
monoclonal antibodies
inject protein into organism, makes B cells, fuses to tumor cell to make "hybridoma", makes more antibody -> target protein
polyclonal antibodies
inject protein into organism, protein activates B cells, plasma B cells make polyclonal, get antiserum
what does slippage during DNA replication cause?
insertions and deletions. happens a lot in repetitive sequences
translation initiation in eukaryotes
involves binding of factors to 5' cap and 3' poly A tail. ribosomes scan 5' UTR for AUG. otherwise, similar to bacteria: start with small subunit, E and A site blocked by eIFs and NO mRNA YET...tRNA-Met brought in before binding occurs ternary complex (3 components) = Met-tRNAi + eIF2-GTP (eIF2 bound to GTP and charged initiator tRNA, which is then delivered to P site) ribosome binding to mRNA involves m7G cap and poly A tail binding proteins
what's true of C-terminal glycine?
it covalently links to lysine
what's true of the genetic code?
it designates the amino acids that are specified by each codon degenerate; most amino acids can be designated by more than 1 codon shared by all life forms non-overlapping and continuous
why might the vaccines we have not work on the omicron variant?
it has 32 mutations in its spike protein, so the antibodies we have as a result of the vaccine may not recognize it when it enters the body
how is translation elongation different in eukaryotes than bacteria?
it's basically the exact same steps, but eEF1A does EF-Tu's job, and eEF2 does EF-G's job. it has similar factors in both that give aminoacyl-tRNAs to ribosomes and other ribosomes along mRNAs
what's true of DNA replication?
it's how DNA is generated, in a semiconservative way; after replication, each daughter DNA gets 1 from parent, and 1 brand new. this is compared to potential mechanisms of conservative (2 parental, 2 new) or dispersive (interspersed regions of old and new) methods of distributing DNA to offspring. can observe semiconservative nature by using N isotopes to differently mark parental and new DNA
what's true of the translation of nonstructural proteins (enzymes) for COVID-19?
it's very different than the translation of its structural and accessory proteins (M, S, E) which are translated from +sgRNA...nonstructural are translated from +gRNA
replisome
low error rate of 1/100000. has proofreading activity (nsp14), helicase (nsp13), SSB (nsp9), polymerase activity (nsp12), processivity factors (nsp7, 8)
2'-O methyltransferase
methylates ribose at 2' position. cap1 and cap2 methylations increase binding of translation initiation factor eIF4E. humans: almost all mRNAs have cap0 and cap1, %50 have cap2. some organisms have cap3 and cap4
miRISC
miRNA induced silencing complex...inhibits mRNA translation, promotes decay
which repair mechanisms are dependent on DNA replication?
mismatch repair of DNA replication errors, translesion synthesis, homologous recombination repair of DNA 2x strand breaks + ATM kinase
example of missense mutation?
missense in Ras gene causes cancer. G12V mutation blocks GTPase activity of Ras, making it permanently active. it's mutated in ~30% of human cancers
what do point mutations in NON-CODING regions alter?
molecular interactions. TFs bind to enhancer, GTFs bind to promoter...mutations affect their abilities to bind, also affecting snRNPs, RBP, RISC
HIV-1 reverse transcriptase (RT)
multi-function enzyme: 1. synthesized negative strand of DNA with viral RNA as template strand 2. hydrolyzes viral positive DNA 3. synthesizes positive strand of DNA with dNTPs, has RNA-dependent DNA polymerase activity, RNaseH activity, and DNA-dependent DNAP activity
common features of tRNAs?
nucleotide sequence, modifications, structure
basics of DNA replication?
occurs in 5' -> 3' direction the energy needed comes from hydrolysis of nucleotides requires DNAP, and primer (not for RNA!)
what's true of the different DNA strands during replication?
one is discontinuous (Okazaki fragments) other is continuous
endocycling
only G and S phases of mitosis
where does A to I editing occur in bacteria?
only at AGG anticodon on tRNA for Arg
E. coli DNA replication initiation
oriC (245 bp) sequence, 2 sections (A/T rich DNA with unwinding elements, and DnaA boxes). can be slow growth (rep starts 1/cell cycle) or fast growth (rep starts before previous round ends)
What DNA sequences are bound by the following proteins?
oriC is bound by DnaA ARSs (autonomously replicating sequences) are bound by ORC in yeast origins are bound by ORC in humans -10 and -35 promoter elements are bound by bacterial sigma factor enhancer elements are bound by eukaryotic transcription factors (TFs) TATA box (promoter element) is bound by TATA-binding protein (TBP)
OGRE
origin G-rich repeat element
ORC
origin recognition complex
how many tRNA genes are there in the human genome?
over 500
how many different modifications are found in tRNAs?
over 90 humans = ~13 mods on average yeast = 7-17 mods
what's true of polar vs. non-polar amino acids?
polar amino acids get modified more often than non-polar ones. they are: serine, threonine, tyrosine, cysteine, lysine, asparagine
what does RNA/RNA base pairing do for translation initiation in bacteria?
positions initiation codon (AUG...methionine, no degeneracy) in the P site. IF3 and IF1 then block the assembly of 50S subunit and mRNA. IF2 bound to GTP brings charged initiator tRNA to P site (makes ternary complex fMet-tRNA + IF2 + GTP). hydrolysis of GTP to GDP then releases IF2 and allows 50S subunit assembly
what does the NF-KB pathway illustrate well?
post-translational events 1. pathogen meets receptor, and undergoes phosphorylation (post-trans mod) to become IKK2, a kinase 2. IKK2 undergoes phosphorylation again to become IKBa, an inhibitor 2a. IKBa can undergo additional ubiquitination, and then proteasomal degradation OR 2b. IKBa can meet up with an NF-KB transcription factor, which then undergoes nuclear translocation to signal inflammatory genes
bacterial cell cycle
pre-replication stage (1 loop) little loop forms in big loop yin-yang loop 2 loops apart, but connected post replication stage (loops split into separating babies) 2 new babies
what binds translation termination codons?
protein release factors. after this, ribosomes get released from mRNA and are recycled for next round
post-translational modification
proteins get changed to shift interactions, localization, function, or stability. there are 5 types: 1. add chemical groups (phosphorylation, methylation, acetylation, hydroxylation) ex: serine -> phosphoserine 2. amino acid modifications (deamidation, disulfide bond formation) ex: cysteine + cysteine -> 2x cysteine with di-S bond 3. cleavage (proteolysis) ex: protease 4. addition of complex molecules (glycosylation, lipidation) ex: sugar-adding enzymes 5. addition of polypeptides (sumoylation, ubiquitination) ex: ubiquitin ligase
release factors
proteins whose structure mimics tRNA. they bind stop codons. GCQ motif of domain 2 performs hydrolysis of ester bond between peptide and tRNA in the neighboring peptidyl-tRNA that's in the P site of the ribosome
what are the 2 ribosomal subunits made up of?
rRNA and proteins
telomeres
repetitive DNA sequences at the end of chromosomes. humans = T2AG3 repeated for 5000-15000 bp. they are not recognized as a break in 2x stranded DNA
telomerase
replicates chromosome ends (telomeres). discovered by Blackburn, Greider, Szostak. lengthens 3' overhang (anneals to 3' end), has reverse transcriptase activity, its RNA hybridizes to make DNA template, and then complementary strand gets replicated
Cdc6
required for loading of helicase, allows Cdt1 to bind to ORC
where does codon recognition happen?
ribosomal small subunit
ATM kinase
serves multiple roles in 2x strand break repair...promotes repair of 2x strand breaks for both NHEJ and HR, and promotes cell cycle arrest and autophagy by getting recruited to cell with MRN. becomes modified post-translation and monomerized, leading to repair/arrest/autophagy
what target proteins to the endoplasmic reticulum (ER)?
signal sequences. function as zip codes to localize proteins, with several different resulting functions: import to ER, retain in ER lumen, import to mitochondria, import to nucleus, or import to peroxisomes
what's true of tRNAs and the codons they pair with?
some tRNAs base pair with more than 1 codon, and not all codons have a corresponding tRNA with perfect base pairing. this is due to wobble base pairing
example of splice site mutation?
splice site mutation in CHD7 gene causes CHARGE syndrome. wildtype: agGGG, but frameshift of nucleotides downstread leads to mutant: aGGA
EF-G
structurally similar to ternary complex
what does DNA replication cause to form in front of replication forks?
supercoils. can be positively or negatively charged, get relaxed by topoisomerases: Top1 = cuts 1 strand, allows controlled rotation, then re-ligates. + or - Top2 = cuts both strands, allows duplex passage, then re-ligates. + or - Top3 = cuts 1 strand, intact strand passes through broken one, re-ligates. only -
3 types of point mutations
synonymous (doesn't change amino acid...degeneracy) missense (changes 1 amino acid into another) nonsense (changes amino acid into stop codon)
what's unique to bacteria regarding base pairing and AUG?
the Shine-Delgarno sequence base pairs with the mRNA and positions AUG in the P site
TRS
transcriptional regulatory sequence
what does ribosome scanning identify?
the initiation codon AUG. scanning of 5' UTR done in 5' -> 3' direction, and 90-95% of the time it starts at the 1st AUG present. the RNA helicase activity of eIF4A allows for scanning
Hayflick limit
the number of times a human cell is capable of dividing into two new cells. the limit for most human cells is approximately 50 divisions
why are the effects of mutations in coding regions smaller than you'd think?
the organization of genetic code makes it tolerant of point mutations. for 8/16 possible amino acids, mutating the 3rd letter doesn't change it. additionally properties of amino acids make them tolerant of mutations (nonpolar/polar, + and - charge)
what's up with chromosome ends and replication?
they can't be replicated by normal replication mechanisms, as there would be a 3' overhang of unreplicated DNA if you did it the normal way. have telomeres instead which get replicated by telomerase
what's true of eukaryotes and miRNAs?
they encode many. humans have 1522, with 50% of mRNA regulated by miRNA...affects cell differentiation and proliferation
what's a key factor of ribosomes that makes the whole system of translation work together?
they have 3 binding sites for tRNAs: E site, P site, A site. they also have an "exit tunnel" where the new polypeptide chain exits
what's true of translation factors?
they interact with both unique and common features of tRNAs
what's true of GTPases?
they're central to translation initiation, elongation, and termination. undergo conformational changes with GTP binding and hydrolysis to GDP...these changes promote the movement of initiation factors
what's true of ubiquitinated proteins?
they're degraded by proteasomes. the ubiquitin-proteasome system is responsible for 80-90% of cellular proteolysis, and the remaining 10-20% is carried out by autophagy. ubiquitin is covalently linked to lysines in target proteins. it has 76 amino acids, 7 of which are lysines (K) for making chains. links to K11 and K48 are involved in protein degradation
what's true of bacterial and eukaryotic ribosomes?
they're functionally analogous. they have both rRNAs, and proteins
what's true about proteins and the first ORF of +sgRNA?
they're generally only produced from the first ORF of +sgRNA
what's true of transcription and translation in bacteria?
they're physically separated from one another
what's true of the DNAP used in bacterial DNA replication?
they're specialized: DNAP 1 = replaces DNA primers/exonuclease DNAP 2 = some replication, exonuclease DNAP 3 = main replication, exonuclease DNAP 4 = translesion synthesis
overview of start and stop of translation in bacteria?
translation of mRNAs with 1 gene (monocistronic...1 protein encoded by 1 mRNA), looks like: 5'PPP -(SD)--AUG--(I...lac repressor)--UAA-OH3' translation of genes in operons (polycistronic...several proteins encoded by 1 mRNA) looks like: 5'PPP-(SD)--AUG--(Z...B-galactosidase)--UAA *break* (SD)--AUG--(Y...permease)--UAA *break* (SD)--AUG--(A...transacetylase)--UAA-OH3' translation of mRNAs in eukaryotes looks like: 5'm7G-AUG--(actin)--UAA-AAAAAAAAAAOH3'
translation
turning RNA into proteins. involves ribosomes, tRNAs, amino acids, mRNAs
Kornberg experiment
used biochemistry to identify DNAP. ground up bacteria, added dNTPs and single stranded DNA, but didn't get no replication. then ground up bacteria, added dNTPs, single stranded DNA, and a PRIMER, and got replication
properties of miRNAs
variable base pairing at 3' end 6-8 nt seed base pairing at 5' end ~21 nt long base pairing leads to mRNA specificity don't perfectly base pair to mRNA...work with several sequences (degeneracy?) assemble with proteins in miRISC
viruses
wants to make copies of themselves. their genomes encode proteins for making more viruses and proteins/ncRNAs that function to block host cell defenses. require host cell factors to transcribe and translate viral mRNAs to make more viruses. 6 stages: attachment, entry, uncoating, genome replication and protein production, assembly, and virion release
how many origins of DNA replication are in eukaryotes?
yeast = 500-1000, A/T rich, called "ARSs" humans = >250,000, A/T rich then CpG region then OGRE genomes are a lot bigger than bacteria, so need more than one place to begin replicating when needed. little bubbles of DNA replication form at each origin, which then eventually converge into 1 big bubble
how many DNAP do eukaryotes have?
~15, but only 3 involved in DNA replication: alpha = initiates DNA replication in conjunction with primase delta = replication of lagging strand epsilon = replication of leading strand telomerase = replication of chromosome ends
