1.11 - Translation Flashcards
Shine-Dalgarno box (bacteria)
consensus sequence that pairs with 16S RNA (part of small ribosomal unit)
role of Shine-Dalgarno box
binding between sequence and 16S RNA brings rest of small ribosomal unit to mRNA
how does Shine-Dalgarno box allow for polycistronic genes in bacteria?
sequence allows for ribosome to bind throughout transcript and to initiate transcriptions
start codon in bacteria
AUG - codes for amino acid methionine
form of methionince required for initiation in bacteria
methionine with a formyl group (fMet) on specialised tRNA
what does the specialised tRNA with fMet bound allow for in bacterial translation?
allows it to bind to middle P-site of ribosome from start (other amino acids required to start at A-site before -> P-site)
what does tRNA binding to the large ribosome allow it to do?
bind to mRNA, tRNA and small ribosomal subunit, creating the ribosome
initiation factors
proteins that are important to ensure translation begins
3 important initiation factors in bacteria (3)
- IF1
- IF2
- IF3
IF1 role
blocks tRNA from entering ribosome subunit
IF2 role
binds specifically to initiator fMet tRNA to bring it to start codon
IF3 role
ensures there is match between codon and anticodon for fMet
when do IF1, IF2 and IF3 dissociate?
upon joining large ribosomal subunit, critical for ensuring correct start to translation
simultaneous transcription/translation in bacteria?
as DNA being transcribed into RNA, ribosomes are able to bind to transcript to start translation
what enables simultaneous transcription/translation in bacteria? (3)
- both take place in the cytoplasm
- they are seeking only the Shine-Dalgarno box for ribosomes to bind
- multiple ribosomes/polyribosomes able to bind at same time to same transcript
how is translation initiated in eukaryotes?
(no Shine-Dalgarno box)
small ribosomal subunit binds to 5’ cap of mRNA, moves along mRNA until it reads AUG codon
difference of methionine in eukaryotes
normal methionine (not fMet) on an indicator tRNA is recruited before assembly of large ribosomal subunit
initiation factors required to ensure mRNA transcript is complete in eukaryotes (2)
- elF4E
- elF4G
elF4E role
replaces 5’ cap protein on the transcript
elF4G role
binds to poly(A) binding proteins and elF4E
when can eukaryotic initiator factors dissociate
once small ribosomal subunit has found first AUG, allowing large ribosomal subunit to bind
where does transcription occur in eukaryotes?
nucleus
where does translation occur in eukaryotes?
cytoplasm
polyribosomes
can bind to mRNA transcript and translate multiple copies of protein
what enables polyribosomes binding to mRNA and translating multiple copies of a protein in eukaryotes?
mRNA tend to form spiral shape as initiation factors and ribosomal subunits can leave and rejoin transcript in close proximity
2 mechanisms microRNA can block transcription (2)
- binding to mRNA transcript and blocking translation from initiating
- degrading mRNA transcript so no translation can occur
three tRNA binding sites in ribosome (3)
- A-site (aminoacyl)
- P-site (peptidyl)
- E-site (exit)
(exist on both small and large ribosomal subunits)
order tRNAs move through ribosome sites
A –> P –> E
where do first amino acid and tRNA in ribosome come from?
initiation in P-site
name of tRNA in P-site
peptidyl-tRNA (tRNA attached to the polypeptide chain)
what does an open ribosomal A-site allow for?
aminoacyl-tRNA able to enter ribosome and bind if anticodon is correct
where does anticodon binding occur in ribosomes?
small ribosomal subunit
when does peptide bond formation occur between new amino acid and polypeptide chain?
when new aminoacyl-tRNA enters A-site
what enzyme mediates joining of new amino acid to polypeptide chain?
peptidyl transferase
peptidyl transferase
RNA enzyme inside large ribosomal subunit, mediates joining of new amino acid to polypeptide chain
peptidyl transferase mediated reaction (3)
- mediates nucleophilic attack from nitrogen terminal of new amino acid into ester bond of tRNA of polypeptide chain
- breaks ester bond between polypeptide chain and peptidyl-tRNA and creates new peptide bond between polypeptide chain and amino acid on aminoacyl-tRNA
- polypeptide chain transferred from peptidyl-tRNA in P-site to amino acid on aminoacyl-tRNA in A-site
what direction are peptide bonds formed?
N (amino) terminal to C (carboxyl) terminal
(polypeptide chains always involve amino acids being added to C-terminal)
ribosomal subunit translocation (4)
- with peptide bond formed/ polypeptide chain transferred, large ribosomal subunit translocated first
- moves tRNA in P and A-site into E and P-site respectively
- small ribosomal subunit moves 3 nucleotides along to pair with large subunit
- leaves A-site empty ready to accept new tRNA with amino acid and for E-site to eject empty tRNA
elongation factors
number of GTPases that act as elongation factors which help enhance cycle of tRNA within ribosome
examples of elongation factors and their roles (2)
- EF-Tu/eEF-1a - catalyses binding of aa-tRNA to A-site
- EF-G/eEF-2 - catalyses translocation of peptidyl-tRNA from P/A to P-site
how does EF-Tu help load A-site?
bound to aminoacyl-tRNA, helps bring it to A-site of ribosome
action of EF-Tu if correct codon-anticodon binding occurs (3)
- ribosome causes EF-Tu to hydrolyse GTP that is bound to it
- GTP hydrolysation into GDP causes conformation change to occur in EF-Tu
- causes EF-Tu to dissociate from tRNA-amino acid complex and leave ribosome
translocation in part occurs due to tRNA molecules having change in their affinities (2)
- old P-site tRNA prefers E-site now it doesnt have polypeptide chain attached
- old A-site tRNA prefers P-site since it has polypeptide chain
how does EF-G help with tRNAs move during subunit translocation? (
- EF-G enters ribosome through A-site, binds to hybrid state to stabilise it, placing it in factor binding centre leading to GTP hydrolysis
- changes conformation of protein causing tRNA in A/P-site hybrid location to be forced out of A-site and only into P-site, in turn forces oldest tRNA out
how does EF-Tu help with proofreading?
- GTP-hydrolysis acts as proofreading to make sure correct tRNA has associated (only correct binding will be correctly placed into ribosome)
- delay between EF-Tu leaving tRNA and tRNA entering A-site, providing second proofreading oppurtunity
what will happen to tRNA if there is no codon-anticodon match
tRNA will preferentially dissociate
stop codons (3)
- UAA
- UAG
- UGA
difference between stop codons and termination sites (2)
- stop codons = translation
- termination sites = transcription
what recognises stop codons?
release factor (not tRNA)
how are release factors an example of molecular mimicry?
structure and charge distribution of protein very similar to that of charged tRNA (allows it to enter ribosome)
how do release factors release the polypeptide chain? (
- binds to ribosome A-site
- forces peptidyl transferase to add water molecules to end of peptide chain instead of amino acid
- causes release of peptide from tRNA in P-site
(with additional proteins, the ribosome disassembles)
