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biob10 lec 18 notes (part 2)

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University of Toronto Scarborough
Biological Sciences
Aarti Ashok

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 wit
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