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Lecture

BIOB10Y3 Lecture Notes - Eukaryotic Initiation Factor, Kozak Consensus Sequence, Start Codon


Department
Biological Sciences
Course Code
BIOB10Y3
Professor
Aarti Ashok

Page:
of 3
mRNAs
mRNAs transcribed from genese by RNA poly II
spliced, capped and polyadenylated
exported to cytosol
Ribosomes
2 subunits (large and small)
o LARGE = 3 rRNAs + proteins
o SMALL = 1 rRNA + proteins
**IMP for exam KNOW difference b/w prokaryotic and eukaryotic subunits **
ribosomes are molecular machines
o require energy from GTP hydrolysis
o the ribosomal RNAs are key components to their function
help select the tRNAs, bind protein factors etc.
the crystal structure of the ribosome (prokaryotic) helps us understand how this machine works
each ribosome has 3 sites for association with tRNAs:
1. A or aminoacyl site
2. P or peptidyl site
3. E or exit site
the anticodon ends of tRNAs contact the small subunit (30S) and therefore this part of the ribosome is involved
in decoding the mRNA.
the amino-acid carrying ends of the tRNAs are found to contact the large subunit (50S) imp role in peptide
bond formation
the interface between the small and large subunit are mostly composed of RNA includes the catalytic site
where peptide bond formation takes place
a long tunnel runs through the core of the large subunit (50S) and the newly synthesized protein comes through
there protected
*We put all 3 types of RNA together for translation:
tRNAs (charged with AA)
rRNAs (ribosomes)
mRNAs (transcript of gene)
TRANSLATION
among the most complex processes that occur in cells!
the process is highly conserved in Pro- and Eukaryotes
o more protein factors involved in eukaryotes
steps in translation: (1) Initiation, (2) Elongation, (3) Termination
(1) INITIATION
o Ribosome first attaches to the mRNA to be translated at a specific “initiation codon” = AUG
o Starting at this AUG, the ribosome will read the triplet codons = reading frame
Step 1
o the small ribosomal subunit binds to the AUG (start) codon
o In prokaryotes, the correct AUG codon is chosen by the presence of an upstream sequence: The Shine-
Dalgarno sequence (-5’-AGGAGG-3’) 5-10 nucleotides before the start
o Shine-Dalgarno sequence is complementary to the 16srRNA of the small ribosomal subunit, thereby
anchoring the ribosome at the right spot
Translational initiation requires initiation factors (IFs) IF1, IF2, IF3 attach to 30S subunit at this stage:
o IF1 : helps the 30S subunit bind the mRNA
o IF2: GTP-binding protein that helps the first tRNA to attach
o IF3: prevents the 50S (large) ribosomal subunit from attaching to the small subunit prematurely
Step 2
o AUG codes for methionine : both prokaryotes and eukaryotes
in bacteria, it is N-formylmethionine
o the initiator tRNA (fMet)binds in a region that will be the ‘P’ site of the ribosome: it interacts with the
AUG on the mRNA as well as IF2
Step 3
o at this stage, IF1 and IF3 are released
o once IF3 comes off, the large subunit now attaches to the complex
o the GTP bound to IF2 is hydrolyzed and a conformational change in ribosome then releases the IF2-GDP
Initiation
in EUKARYOTES
o Kozak sequence on mRNA: 5-'ACCAUGC-3'
o For initiation to occur in eukaryotes, several eukaryotic initiation factors (eIFs) must bind to the 40S
(small) subunit, along with the tRNA for methionine (AUG) and this whole complex then interacts with
the mRNA starting at the 5’ cap. It then scans along the mRNA until it finds the AUG start. translation
initiation complex
(2) ELONGATION
Step 1
o the next aminoacyl-tRNA come into the empty A site of the assembled initiating ribosome
o this tRNA first has to bind an elongation factor EF-Tu
EF-Tu is bound to GTP
o any tRNA-EF-Tu-GTP complex can “sample” the A site, but only the one whose anticodon matches the
next codon on the mRNA would be allowed to bind causes a conformational change in ribosome
o once the right tRNA is bound in the A site, GTP hydrolysis occurs and EF-Tu-GDP is released from the
ribosome, leaving the right tRNA behind
o EF-Tu-GDP is regenerated into EF-Tu-GTP by a factor known as EF-Ts
Step 2
o now, the 2 amino acids attached to the tRNAs in the A and P sites are ready to react with each other
o amino acid attached to tRNA in P site becomes linked by a peptide bond to the amino acid attached to
the tRNA in the A site
=peptide bond formation
requires no energy
catalyzed by peptidyl transferase (ribozyme part of 23s rRNA)
o so, A site tRNA has a dipeptide attached to it and P site tRNA is deacylated
Step 3
o the next step involves movement of the ribosome along the mRNA in the 5’ 3’ direction
=translocation
requires the activity of EF-G
GTP hydrolysis occurs
o the deacylated tRNA is moved into the E site and the tRNA with the dipeptide now moves into the P site
Step 4
o the deacylated tRNA now leaves the ribosome entirely from the E site
o A site is now open for the next tRNA to enter the ribosome and lines up with the next codon
o once it has bound, then the AA attached to that tRNA will form a peptide bond with the dipeptide
attached to the P site tRNA
P site tRNA is deacylated
A site tRNA will move into P site when ribosome translocates
Elongation
in EUKARYOTES
o EF1α = EF-Tu in prokaryotes
o EF2 = EF-G in prokaryotes
o a conformational change in the ribosome here after EF1α-GTP hydrolysis, puts the 3’ ends of the tRNAs
in close proximity for peptide bond formation to occur
(3) TERMINATION
o when a ribosome reaches a UAA, UAG or UGA codon, there is no corresponding tRNA available
o instead the ribosome will stop elongation and release the polypeptide chain built thus far
ester bond linking the peptide chain to the tRNA in P site cleaved by ribozymes
similar to those that catalyze peptide bond formation
o release factors are required for this:
RF1 recognizes UAA and UAG
RF2 recognizes UAA and UGA
RF3 increases activity of other factors -is a GTPase
o once termination is complete, ribosome disassembles into small and large subunits next round of
translation can begin
Termination
in EUKARYOTES
o (eukaryotic release factor) eRF1 and eRF3 recognize all stop codons
o eRF3 is the GTPase (like RF3 in prokaryotes)
Translation: Polyribosomes
many ribosomes are seen associated with any given mRNA in the cell = polyribosomes or polysomes
each one has attached at AUG site and then started translating from that point
o once one ribosome leaves the AUG start site, another can assemble there and so on