Translation Flashcards
Is translation a reversible process?
Generally, no
Why is it said that the genetic code is mostly degenerate?
AA have many RNA codons coding for them. Not readily reversible
What is the difference between nonsense, silent, and missense mutations?
Nonsense: pt. mut changes codon from AA to a termination seq.
Silent: pt mut. But doesnt change codon recognition
Missense: pt mut. Changes one AA to another
What is between the 5’ cap and the start codon?
5’UTR
What is the universal start codon?
AUG - M/Met/Methionine
What are the stop codons for protein translation?
UGA
UAA
UAG
Where is the poly(A) tail added? How many nucleotides is it composed of?
In the nucleus, about 200
If a base is deleted from the genetic code, what type of translational error will result?
Frameshift
What type of RNAs read protein code?
TRNAs
At what end of tRNAs is the AA attached to?
3’
What is the purpose of the anticodon loop?
Codon recognition
What enzymes recognize tRNAs and add the appropriate amino acid? IS this an ATP required step?
Aminoacyl tRNA synthetases
ATP required
How many types of aminoacyl tRNA synthetases are there?
20.
How many active sites do AAtRNAS have? What are their purposes?
2
One for synthesizing and one for editing
How do we classify ribosomes?
Ribozyme - peptide formation without contribution of a protein.
What is the 80S eukaryotic ribosome composed of?
60S and 40S
What are the three tRNA binding sites on ribosomes?
A (aminoacyl site) -> initial tRNA binding
P (peptidyl site -> peptide binding site
E (exit) -> tRNA release
What are the three phases of mRNA translation?
- Initiation
- Elongation
- Termination.
(You can have multiple ribosomes on a single RNA molecule)
What tRNA molecule does the 40S subunit bind with to produce a pre-intiation complex?
Initiator tRNA.
What is the initiator tRNA usually associated with?
Binds methionine and eIF2
What is eIF2 usually associated with?
GTP. GTP hydrolysis acts as a switch to start up sequence.
What does eIF4E associate with?
5’ cap of mRNA
What does eIF4g associate with?
/eIF4E creates a sort of scaffolding complex
What is the only step that requires ATP during mRNA translation?
When the initiator moves along mRNA it requires helicase activity to keep going looking for an AUG. This requires ATP
What is step 2 of the initiation sequence of translation?
Scanning mRNA for AUG codon
What is step 3 of initiation sequence of translation?
Large subunit joining
What happens when large ribosomal subunit binds?
GTP hydrolysis to GDP and eIF2 and other initiation factors dissociate.
What is the first step of elongation in protein translation?
Delivery of aa-tRNA to A site and E site release.
Aa-tRNA delivered with eEF1A bound with GTP.
How does aatRNA bind to A site?
EEF1A associated with GTP guide molecule and GTP hydrolysis binds it to A site while kicking of eEF1A
What is second step of elongation in protein translation?
GTP hydrolysis and eEF1A release
What happens if the wrong aatRNA is delivered?
Preferentially dissociate.
What is eEF2’s role in protein translation?
Catalyzes translocation. Signals to ribosome to move on to next codon
What is the 3rd step of elongation in translation?
EEF2 binding to catalyze translocation
What is the 4th step of translational elongation?
GTP that was bound with eEF2 gets hydrolyzed and dissociates. Opens the A site back up.
At what step in translational elongation does the E site release?
1st step. With aa-tRNA delivery via eEF1A and GTP
What tRNAs does eEF1A not bind to?
Initiator tRNA + selecocysteine tRNA
Binds all others
Are there any terminating tRNAs? How does protein translation terminate?
No.
Protein -> eRF1 (releasing factor)
Bacteria have 2 of these RF.
Comes in at stop codons:
- UAG
- UGA
- UAA
Does bacterial mRNA have a 5’ cap?
No, they are polycistronic and translation happening immediately after transcription.
What initiates bacterial translation?
Shin-Dalgarno sequence (upstream of codon); directly BP with ribosomal RNA.
Compare and contrast Eukaryotic and Prokaryotic translational methods:
Eukaryotic: Prokaryotic
- Monocistronic mRNA. Polycistronic
- 40S+60S->80S ribosome. 30+50->70S
- EIF4e (5’ cap bound). No counter
- EIF2 (binds initiator tRNA w/gtp). IF2
- EIF4G (Binds to eIF4E, scaffold) No counter
- EEF1A (deliver AAtRNA, kicks out E). EFTu
- EEF2 (translocation (frees up A site). EFG
How is translation post-transcriptionally regulated?
- RNA editing (rare)
- MiRNAs
- Iron storage
- Poor nutrition
- Hypoxia
What is an example of mRNA editing?
MRNA editing happens post-transcription, but pre-translational.
ApoB is made in liver and intestine.
Intestine needs a shorter form of the protein.
ApoB mRNA is edited to produce a premature stop codon (via a nonsense mutation)
What are miRNAs?
Short RNA molecules that BP w/ mRNAs and regulate translation (typically during development)
How do miRNAs inhibit translation?
BP with 3’UTR region and create RISC complexes thought to physically impede translation
What diz are correlated with miRNA function?
- Cancers (breast,lung)
- Viral diz (HCV, HIV)
- Immune related diz
- Neurodegenative diz
How is iron homeostasis regulated?
Iron sensing protein (IRP)
What is the iron regulatory element? How does it work in regards to iron levels?
Stem-loop sequence of mRNA structure.
Iron regulatory protein binds with there is no iron ->prohibiting translation
If there is iron present, IRP cannot bind with stem-loop IRE and translation proceeds.
Gene is on ferritin mRNA
When there is a shortage of nutritional AA, what happens to protein syn?
Down regulated
How does eIF2 become phosphyraled?
- [aa] decreased
- Cellular stress
- Immune response
- Unfolded proteins
Why are guanine exchange factors (GEF) important?
After GTP gets hydrolyzed to GDP, need something to exchange it. Back.
GEFs req. b/c affinity for GDP is much higher than that GTP
What is the GEN for eIF2?
EIF2B
What happens when eIF2 gets phosphorylated during stressful events?
This phosporylated eIF2 sequesters eIF2b and forms an inactive complex
What does mTOR stand for? What does it do?
MTOR- mammalian target of rapamycin.
Signaling pathway that represses translation in response to hypoxia thru eIF4E regulation.
Cellular rheostat. (Upregs in growth, down in stress)
In normoxia and growth, how does mTOR regulate eIF4E?
It does not directly regulate on top of eIF4E. Instead presence of mTOR phosphorylates 4EBP-1 which prevents them from binding to eIF4E
What happens when hypoxic cell conditions are introduced into the cell (in terms of mTOR)>?
Less mTOR is produced, which reduces 4EBP-1 inactivation. Allows 4EBP-1 to bind with eIF4E and inhibit translation.
What is the 21st AA? How is it produced?
Selenocysteine (essential)
Enzymatically converted from serine.
Incorporates via modified UGA (stop) codon
What enzymes that use selenocysteine are required for thyroid hormone synthesis?
Deiodinases
What is the elongation factor that recognizes Sec-tRNA?
What is the tRNA that recognizes special UGA codons?
What is the SECIS element?
What is Secis BP 2 (SBP2)?
- EEFSec
- Sec-tRNA sec
- 3’ UTR rna seq. Required for sec incorporation
- Binds secis region and helps recruit to get sec in at the specialized stop codon
When can a single mRNA encode for more than one protein?
Prokaryotes
Also is ribosome is frameshifting
How does a ribosome frame shift?
- Slippery site
2. Pseudoknots.
How does the diptheria toxin work?
Modifies elongation factor responsible for translocation (eEF2). (3rd step of elongation)
