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67 Cards in this Set
- Front
- Back
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Where does the AA attach to the tRNA?
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the carboxyl part of the AA attaches to the 3' A to either the 3' or 2' OH group there.
Forms an ester bond there. |
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What is the enzyme that catalyzes putting an AA on tRNA?
How many different types of this enzyme are there? |
aminoacyl-tRNA synthetase
Each AA has a unique aminoacyl-tRNA synthetase and it's own unique tRNA too. |
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How important is the fidelity of protein synthesis?
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VERY IMPORTANT. Cannot exceed ~10to the -4 error frequency. The bigger the protein the less errors can occur.
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When a new AA is brought to the ribosome, where is the peptide bond formed?
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AA #1 amino group attaches to the carboxy group of the incoming AA#2.
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What is the 5' end of a tRNA?
3' end? What is the anti-codon for yeast alanyl tRNA? |
phos'd G
CCA with an OH IGC which codes for GCC= Alanine |
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How can there be variation in tRNA's?
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modified nucleosides
methyl inosine mI dihydrouridine UH2 ribothymidine T pseudouridine (psi) methyguanosine mG dimethylguanosine m2G inosine I |
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describe the structure of tRNA's.
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single chains of 73-93 ribonucleotides
has different modifiend bases (like methyl G or I or etc) overall cloverleaf shape 50% of bases form double helices to make the L shape conformation anti-codon at one end and AA attachment site at the other end. |
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What is the purpose of the variations among the modified ribonucleotides?
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increases hydrophobicity
prevents certain base pairing alters codon recognitions |
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In order from 5' to 3' end, what are the 5 groups of bases that are not paired?
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5' phos'd G
DHU loop (several UH2 residues) Anti-codon loop Extra arm TYC loop (ribothymine pseudouracil cytosine loop) 3'CCA acceptor stem |
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Where is the amino acid linked to the tRNA?
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the carboxyl end of AA attaches to 2' or 3' OH of ribose (creating an ester bond) which is on the 3' end of the CCA cap of tRNA
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What is a charged, activated tRNA called?
What enzyme activates them? |
aminoacyl-tRNA
aminoacyl-tRNA synthetases |
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How do AA get put on a tRNA?
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aminoacyl-tRNA synthetases use 2 ATP to make a mixed anhydride with adenylate
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Describe how threonyl-tRNA is able to accurately incorporate only Thr when Valine is so similar?
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Thr-tRNA synthetase has a zinc ion coordinated with 2 His and a Cys. The zinc holds Thr with its OH to Thr's amine.
Another residue, a Asp, bonds with the Thr OH Valine has a side chain methyl which cannot interact with zinc or Asp |
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Ser, similar to Thr, has a side OH that CAN interact with the zinc ion in Thr-tRNA synthetase. How does the enzyme differentiate to be accurate between these 2 AA?
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Thr synthetases have an editing site. The CCA-arm swings about 20 A away.
The editing site will reject AA's larger than the target and thus not hydrolyze them. Because Ser is smaller than Thr it fits into editing site and gets zapped |
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When it's time to put the aminoacyl adenylate on the tRNA what does the synthetase interact with?
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Some tRNA's interact only with anticodon, some form complexes with several sites on the tRNA like the acceptor arm and anticodon
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Explain how experiments with E. coli can show how some synthetases don't even need the anticodon region
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E. coli tRNA cys is different from tRNA ala in 40 positions and also in 3:70 it has CG
With mutagenesis experiments changing the CG to non pairing GU makes tRNA cys recognized by tRNA ala synthetase also, these experiments showed that only 24 residues and a hairpin loop were all that was needed for tRNA ala synthetase to recognize the tRNA. |
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What are the differences between class I and class II aminoacyl-tRNA synthetase
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They bind to different faces of tRNA and CCA arm changes conformations to accomodate this
Class I : CCA arm is in hairpin acylate 2' OH of terminal A most are monomeric Class II: CCA arm is helical acylate 3' OH of terminal A (expcept Phe-tRNA) most are dimeric |
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What are the 2 subunits of the E. coli ribosome and what do they each contain?
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30S
-- S1-21 proteins (S20=L26) -- 16S rRNA 50S -- L1-34 proteins -- 5S and 23S rRNA --2 copies of L7 and L12 proteins |
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How is the 30S subunit formed in E. coli ribosome?
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from rRNA's
5S, 16S, 23S These rRNA's provide architecture and folding into defined structures with short duplex regions |
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In ribosomes, even though there are a lot of varying proteins in there, what is the meat and potatoes of the active site?
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All ribosomes.
Hence you avoid the chicken and the egg debate. The extra proteins are add ons that fine tune but RNA does all the heavy lifting in protein syn. |
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What is a pulse labeling study and what does it prove about how proteins are synthesized?
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Radioactive labeling of reticulocytes as they were synthesizing hemoglobin.
Different lengths of time samples showed that the earlier examined hemoglobin had only carboxy ends that had incorporated the label and then the later examined hemo had incorporated the label closer to amino terminus as well. So protein gets built with amino terminus not able to include label yet because it happens first, but since the carboxy end gets added later it has more labeled stuff in it. |
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Which way direction does protein synthesis proceed in?
Why is this significant? |
From amino to carboxy direction
5' -->3' As you know, in pro's translation and transcription are coupled and since protein syn proceeds in same direction as mRNA is being made, translation can occur as the mRNA is still being finished. |
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What is a polyribosome?
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a group of ribosomes on an mRNA strand translating away.
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What is the code for methionine and why is this significant?
What are some other codes of importance for the same reason? |
AUG
This is the initiator codon in pro's. GUG is for Val (less frequent) UUG is for Leu (rarely) |
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Where does protein syn translation occur on the mRNA?
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more than 25 nucleotides away from the 5' end
Usually intiated by AUG (met) |
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What is the Shine-Dalgarno sequence in mRNA?
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purine rich sequence centered 10 nucleotides on 5' end of initiator AUG (or GUG or UUG)
It binds with the 3' end of 16S part of the 30S subunit Mutating this really messes up the initiation of protein syn |
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What are the 2 important features of protein synthesis initiation?
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initiator mRNA codon matching (AUG, GUG or UUG) with tRNA anti-codon
Shine-Dalgarno sequence which interacts with 3' end of 16S part |
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What is the modified initiator codon in E. coli proteins?
How does this special guy impact its matching tRNA? |
fMet
formylated Methionine a special initiator tRNA called tRNAf (as opposed to tRNAm) brings this fMet to the ribosome. |
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When does the modification of Met occur in the initiation of E. coli proteins?
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Once Met is on tRNAf (has to be this special tRNA--doesn't work with tRNAm) a transformylase gets a formyl from N10 formylTHF adds it to tRNAf to make fmet tRNAf
tRNAf uses the same synthetase as regular tRNAm |
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What are the 3 tRNA binding sites between the 30S and 50S subunits?
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A=aminoacyl site
P=peptidyl site E=exit site |
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Which 2 of the 3 tRNA binding sites in the ribosome are bound to the mRNA on the 30S side?
What are they bound with? |
A site
P site E site is unbound to mRNA Their anticodon site base pairs with the codon on mRNA |
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What interacts with the 50S subunit on the tRNA?
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the acceptor stem (CCA part attached to amino acid)
the peptide bond is formed between amino acids and the newly formed protein starts emerging out the tunnel in the back of the ribosome |
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What does the complete, ready to make some proteins 70S ribosome consist of?
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fMet-tRNAf bound to AUG while also interacting with Shine-delgarno region in P site.
Now the initiator is locked and loaded, bridging the 30S and 50S subunits and another aminoacyl tRNA can come in to match the next codon. |
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Where on the 50S subunit is the peptide bond between AA's formed?
How does it happen? |
23S rRNA of 50S subunit== peptidyl transferase center
amino group of the incoming aminoacyl tRNA in the A site attacks the ester bond between fMet (or whatever proceeding AA) and tRNA that is in the P site the intermediate is stabilized by surrounding bases peptidyl transferase center |
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Which site does the attack of the incoming amino tRNA on the 50S subunit?
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P site
translocates so that it wiggles its way to the E site |
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What is the protein enzyme that moves the mRNA so that the new codon is in the A spot?
How does it do this? |
elongation factor G (EF-G)
uses GTP hydrolysis to translocate the mRNA |
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Where is the tunnel that the growing peptide uses as it gets bigger?
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Behind the P site on the 50S subunit. This is where the peptide chain stays.
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Does the amino acid attached to tRNA interact in any way to effect which AA gets added next?
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No, only the codon and anti-codon participate in placing the next AA on the peptide chain.
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What is the wobble hypothesis?
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certain tRNA's recognize more than 1 codon
So the 5' end of the anti-codon (which pairs up with the 3' end of the codon) has more spatial freedom to pair differently I can pair with U, C, or A so there's wobble room |
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What are 2 generalizations regarding the codon: anti-codon interactions that the wobble hypothesis tells us?
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First 2 bases of codon do strict watson-crick base pairing. They have distinct tRNA's
First base of the anti-codon determines whether it will recognize 1, 2 or 3 different codons. |
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Why is inosine so frequently found in anticodon's?
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Because it allows for the recognition of 3 different codon's by the tRNA
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What are the 3 universally conserved base pairs in the 16S subunit?
What do they do? |
A-1492 (colombus sailed the ocean blue
A-1493 (a year later he sailed back G-530 (through the galapagos with only 530 men left) Each conserved residue forms a H bond with both the codon and anti-codon to ensure a proper fit. Like a check to ensure the 1st 2 codon bases are paired up right. No such check for the 3rd base in the codon. |
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What are protein initiation factors?
What are their names? |
They bring mRNA and fMet-tRNAf to the ribosome
IF1 IF2 IF3 |
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What does IF 3 do?
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prevents premature 30S from joining to 50S
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What does IF 1 do?
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binds near the A site and directs fMet-tRNAf to P site
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What does IF2-GTP-initiator do?
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binds mRNA and 30S subunit together.
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What does the 30S initiation complex consist of ?
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IF 1 bound to 30S near A site
IF3 bound to 30S to block premature binding with 50S IF 2-GTP bound to fMet-tRNAf (glues 30S and 50S together eventually) mRNA and fMet-tRNAf |
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What is elongation factor Tu (EF-Tu)?
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The protein that gets the aminoacylated tRNA and brings it to the ribosome A site.
binds in GTP form and never binds fMet-tRNAf protects the aminoacyltRNA ester bond |
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What are the 2 things EF-Tu does as it is bringing the next aminoacyl tRNA to the ribosome?
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protects the ester bond of the tRNA
hydrolyzes GTP bond AFTER a proper codon: anti-codon interaction to add to fidelity. |
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What is elongation factor Ts (EF-Ts)?
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It pries the useless GDP from the EF-tu's hands and resets it for it to go get another AAtRNA.
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Which is stronger, a peptide bond or a ester bond?
What does this mean thermodynamically in peptide bond formation? |
peptide bond is more energetically favorable so the peptide bond formed in protein syn is thermodynamically spontaneous. However, GTP has to be used for translocase to do it's thing and shift the mRNA down
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What is EF-G or translocase?
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an enzyme that utilizes GTP to enhance translocation of mRNA to reveal the next codon.
binds near site A and hangs with 23S rRNA of 50S subunit GTP hydrolysis causes a conformational change and move the strand |
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What are the termination codons?
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UAA
UGA UAG |
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What does RF1 recognize?
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UAA or UAG
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What does RF2 recognize?
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UAA or UGA
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What does RF3 do?
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mediates RF1 and RF2 and their interactions with the ribosome
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Explain how RF1 and RF2 work.
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they bind to stop codon and act as a bridge to the 50S peptidyl transfer center where something happens (we're not sure what) to release the peptide by hydrolyzing the ester bond
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What is the ribosome release factor?
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RRF and EF-G disassemble 70S ribosome in GTP dependent fashion
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How is a Euk ribosome different from a Pro ribosome?
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Pro
30S + 50S = 70S 30S-->16S rRNA 50S--> 5S, 23S rRNA Euk 60S + 40S = 80S particle 40S-->18S 60S--> 5S, 28S, 5.8 rRNA |
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What is the initiator tRNA in Euk?
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Met-tRNAi
Met is the first AA AUG always first codon |
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Is there a shine-daglarno region in euk?
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No
Initiaiton with Met-tRNAi starts at the 5' mRNA cap and the Met-tRNAi scans in 3' direction until it finds AUG |
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What are eIF's?
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Some of the myriad of more complex Eukaryotic Initiation Factors.
eIF-4E binds to the 7methylguanosine cap eIF-2:GTP brings the Met-tRNAi to the ribosome |
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How does Eukaryotic mRNA become circular?
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eIF-4E which binds to the 7methylguanosine cap also binds to the poly AAA tail
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What are the other proteins involved in making Euk mRNA circular?
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eIF-4E
eIF-4G PABPI (poly AAA binding protein) |
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What is EF-1a?
EF-1By? |
EF-1a is similar to Pro EF-Tu. It is an elongation factor that brings aminoacyl-tRNA to the A site
EF-1By is similar to pro EF-Ts which pries the useless GDP and resets it to useable GTP |
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What is the translocation enzyme protein in EUK?
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EF-2
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What is the release factor in EUK?
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eRF-1
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