Week 10 - Translation Pt I Flashcards
elF4F is a heterotrimer consisting of what?
elF4G - scaffold protein eiF4E - binds the 5’ methyl cap elF4A - helicase that unwinds the 5’ structure elF4B binds 3’ poly (A)
What does mRNA bind to?
elF4G, elF3 and 40S subunit
What enables scanning?
elF1 and elF1A
What induces GTP hydrolysis by elF2?
elF5
What mediates joining or 60S subunit?
elF5B
What are the two stages of ending translation?
Termination reaction and post-termination reaction
RF1
Bacterial release factor that recognises UAA and UAG as signals to terminate the polypeptide translation
RF2
The bacterial release factor that recognises UAA and UGA as signals to terminate polypeptide translation
What is the ribosome recycling factor?
RRF
Where does protein synthesis occur?
Cytoplasm
What is mRNA?
Carries the genetic information
What is tRNA ?
Deciphers the codon
What is ribosomal RNA?
Associates with a set of proteins to form ribosomes
Why does tRNA contain non-standard bases?
Modification after transcription
What is the 3’ end of tRNA?
Amino acid attachment site Bind covalently Always CCA
What is anticodon?
Site of complimentary base pairing with mRNA unique for each species of tRNA
What does a combination of 4 bases give?
64 different codons for 20 amino acids
What does ribosome bind with?
A ribosome bind mRNA and tRNA
What are ribosomes?
Ribosomes are ribonucleoprotein particle comprising 2 subunits Each subunit contain a major rRNA(s) and a number of small proteins
In bacteria what does the 70S ribosomal subunits consist of?
Large: 50S (23S + 5S) Small: 30S (16S)
In eukaryotes what does the 80S ribosomal subunit consist of?
Large: 60S (28S + 5.8S + 5S) Small: 40S (18S)
How many binding sites do ribosomes have?
The ribosome has 3 tRNA-bonding sites. A, P and E
What occurs in A site?
An aminoacyl-TRNA enter the A site
What occurs in P site?
Peptidyl-tRNA ( a tRNA carrying the nascent polypeptide chain) is bound to P site
What occurs in E site?
Deacylated TRNA (lacking any amino acid) exit via E site
I’m brief, what is the first stage of translation?
Before peptide formation, peptide tRNA occupies the P site, and aminoacy-tRNA occupies A site
In brief, what is 2nd stage of translation?
Peptide bond formation. Polypeptide is transferred from peptide is tRNA in P site to aminoacyl-tRNA I’m A site
In brief, what is the 3rd stage of translation?
Translocation moves ribosome one codon, places peptidyl tRNA in P site, deacylated tRNA leave via E site and A site is emptied for the next AA-tRNA
What is accuracy of translation controlled by?
Specific mechanisms at each stage. Error rates differ at each stage of gene expression
Give examples of some errors that may occur in translation?
Wrong base frameshift Wrong tRNA Wrong AA Wrong aminoacyl-tRNA
What does bacteria need to start initiation?
30 subunits and accessory factors (initiation factors)
what is the ribosome-binding site
A sequence on bacterial mRNA that includes an initiation codon that is bound by a 30S subunit in the initiation phase of polypeptide translation
Is initiation of translation a function of intact ribosomes?
No it is undertaken by the separate subunits, which reassociate during the initiation reaction
When does recognition of mRNA occur ?
Small subunit bond to form an initiation complex at ribosome binding site
In bacteria, what does initiation of translation require ?
Initiation of translation require separate 30S and 50S subunits
What initiation factor are needed in bacterial initiation?
Initiation factors (IF-1, IF-3 and IF-3) which bind to 30S subunit, are also required
What is the function of IF-3?
Stabilises the free 30S subunits Inhibit premature binding of 50S subunit It enable 30S subunit to bind initiation site Check the accuracy of recognition of the first amino acids-tRNA
What is the function of IF-2?
Bind a special initiator tRNA and controls its entry into the ribosome
What is the function of IF-1?
Binds in the vicinity of A site Prevents aminoacyl-tRNA from entering
Before 50s subunit joins, what must occur first?
A 30S subunit carrying initiation factors Bind to an initiation site on mRNA to form an initiation complex
What must occur in order for 50s subunit to join the 30s subunit?
IF-3 must be released to allow 50S subunit to join the 30S-mRNA complex
What is the initiation site on bacterial mRNA ?
An initiation site on bacterial mRNA consist of AUG initiation codon
What precedes 10 bases before it?
Shine-Dalgarno polypurine hexamer
What is shine-dalgarno sequence ?
Polypurine sequence is AGGAGG centred about 10bp before the AUG initiation codon on bacterial mRNA
What is complementary to Shine-Dalgarno sequence?
Complementary to the sequence at 3’end of 16S rRNA during initiation
Translation starts by what A.A?
Translation starts with a methionine amino acid usually coded by AUG
T or F: the same methionine tRNAs are involved in initiation and elongation
False: different methionine tRNAs are involved in initiation and elongation
What is N-formal-methionyl-tRNA (tRNAfmet) ?
The aminoacyl-tRNA that initiates bacterial polypeptide translation The amino group of methionine is formylated
What is tRNAmMet?
The bacterial tRNA that inserts methionine at internal AUG codons
What are the unique features of initiator tRNA?
NH2 group of methionine - formylated. Bases at the last position of stem are unpaired 3 G-C base pairs are required to allow tRNAfMet to be inserted directly into P site
What tRNA enters the 30S?
Only tRNAfMet enters partial P site on 30S subunit bound to mRNA
What tRNAs enter the 70S?
Only as-tRNA enter A site on complete 70S ribosome
What occurs first in initiation process in bacteria?
IF-1 and IF-2 bind the 30S
What occurs 2nd In the initiation process in bacteria?
IF-2-GTP bind the initiator fMet-tRNAf. This allows it to enter the partial P sure in the 30S subunit
What occurs last in the initiation process in bacteria?
IF-3 leaves and 50s subunits join
What occurs first in initiation in Eukaryotes ?
Eukaryotic 40S ribosomal subunit bind to the 5’ end of mRNA (methylated cap)
What occurs 2nd in initiation in eukaryotes?
40s scan mRNA and migrates to initiation site (AUG) + Kozak sequence
What is the initiation site in eukaryotes?
A eukaryotic initiation site consists of a ten-nucleotide sequence that include an AUG codon (Kozak sequence)
What occurs last in the initiation process in eukaryotes?
60S ribosomal subunit join the complex at initation site
what happens if leader is long?
subunits May form queue
What is IRES ?
A eukaryotic messenger bra sequence that allows a ribosome to initiate polypeptide translation without migrating from 5’end
How many IF in eukaryotes?
At least 12
1st stage of initation pathway in eukaryotes
Ternary complex formation elF2 bind the initiator Met-tRNAi and GTP forming ternary complex that binds to the 40S subunit before it associates with mRNA
2nd stage of initiation pathway in euk.
43S preinitation complex formation 40S subunits assisted by elF1A, elF1 and elF3 interact with complex elF5 bind to 30S subunit elF1, elF3 and elF5 and ternary complex might interact to form multi factor complex that bind to 40S subunit as single entity
3rd stage of initiation pathway in euk.
mRNA activation Eukaryotic mRNA passes the nuclear pre into the cytoplasm as a structure molecule coated with polypeptides mRNA is activated by removing secondary structures and proteins from 5’ end before translation Cap binding process f
4th stage
mRNA entry into preinitiation complex elF4G subunit interact with elF3. Recruit 5’end of mRNA to 43S preinitiation complex Structural changes take place when elF1A and elF1 bind to 40S subunit
5th stage
5’-3’ scanning to detect the initiation codon 43S complex moves along mRNA in a 5’-3’ direction ATP dependent movement continues until the AUg initiation codon on mRNA is aligned with anti-codon in the initatior tRNA elF1 and elF1A both contribute to search for initiation codon
6th stage
Initiation codon recognition Hydrolysis of elF2- bound GTP and Pi release elF5 stimulates elF2 to convert bound GTP to GDP and PI elF1 which usually prevents pi escape is displaced from P site when met-tRNAi bind to AUG.
Escape of Pi triggers conformational change Convert open mRNA entry channel into a closed channel 48S IC can join 60S subunit
7th stage
60S subunit joining and IF displacement elF5B.GTP facilitates the joining of 60s subunit to the 48S preinitiation complex elF1, elF2.GDP, elF5 are elevated during joining
8th stage
Hydrolysis of elF5B-bound GTP and release of elF5B and elF1A elF1A and elF5B.GDP are released after GTP bound is hydrolysed release of elF1A from 80S complex frees the A site to participate in elongation process
What is elF2 ?
GTPasee
What is elF2B?
Exchange factor
What does elF2 consist of?
alpha and beta subunits
What does elF4b?
Stimulates elF4a helicase
What does PAPB do?
Binds 3’ poly (A)
What does elF1 and elF1A enable?
Scanning
What does elF5 induce?
GTP hydrolysis by elF2
What does elF5b mediate?
Joining of 60S subunit
What elFs are involved in pre-initiation complex?
elF2, elF3, elF1 and elF1A
What elFs are involved in cap-binding?
ElF4A, B, E and G
What elFs are I cocked in 48S complex ?
elF2, elF3, elF1 and 1A, elF4A+B+F
What is elF4F?
Heterotrimer consisting of; eif4g (scaffold proteins) eif4e (binds the 5’ methyl cap) eif4a (a helicase that unwinds the 5’ structure)
What is EF-Tu?
Monomeric G protein whose active form binds to amino acid tRNA
What is EF-T?
Guaninine nucleotide exchange factor Required to mediate the replacement of GDP by GTP