BIO206H5 Lecture Notes - Lecture 15: Chief Operating Officer, Gif, Transferase

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31 Oct 2015
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BIO 206 Lecture October 20, 2014-10-20
Translation: Elongation
3 stages
- stage 1: binding
orequires successful recognition in order to move to stage 2
- stage 2: transpeptidation
onew bond is formed from peptide change and amino acid on charged
tRNA
ogrowing polypeptide change is covalently rearranged onto a site tRNA
- stage 3: translocation
orequires ratchet like movement of large subunit to change conformation in
order to move along mRNA exactly 3 nucleotide (1 codon)  new codon is
positioned into a-site
Chain elongation-binding
3 sequential steps
Step 1: binding
- EF-Tu and aa-tRNAaa and GTP formed
- Complex binds at a-site
- GTP  GDP, EF-Tu released
- aatRNA remains bounds
- tRNA molecule in cell is not ALONE IN CELL
othey are in complex with a protein called EF-Tu
othis is actual substrate for the ribosome
oit is a aminoacyl-charged tRNA
othe EF-Tu is also in complex with GTP
oit allows to contain its own energy source
othere is specific site which allows EF-Tu to be binded on
ribosome
oallows tRNA be bounded onto a-site
- EF-Tu is single
polypeptide made
up of 3 distinct
domains
- Compactly
arranged in GTP
complex state
- Domains are
globular in nature
which fold
independently of
eachother
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- GTP molecule is bound in domain 1
- H-bond network, hydrophibic, Van-der walls  allow for tRNA binding site on EF-
Tu protein  binds to acceptor strand of tRNA
- THIS IS SUBSTRATE OF RIBOSOME
- Switch helix is important part of EF-Tu
oIt is involved in mechanism which allows release of tRNA
oIn GTP bound state  only conformation which can accept charged tRNA
oHydrolyse allows for releasing of tRNA
oWhen GTP hydrolyses into GDP  rearrangement of 3 domains of EF-Tu 
changes relationship of forces which hold tRNA molecule
oAll binding mechanisms such as H-bond are disrupted
oEF-Tu has high binding affinity in GTP form, but low binding affinity in
GDP
EF-Tu/aa-tRNA/GTP: substrate
- part of bacterial ribosomal complex: binds
tRNA
GTP bound
- “latch” closed
- tRNA bound
GTP GDP
- “latch” opens
- tRNA released
- hydrolysis causes change in shape of switch
helix
GDP released (EF-Ts)
- ready for new GTP and charged tRNA
WHAT RIBOSOME SUBSTRATE MOVIE
- this shows small subunit of ribosome
and associated complexes
- light blue structure is EF-Tu bound to
surface patch on ribosome
- surface patch allows ribosome to
recognize EF-Tu
- binding results that tRNA molecule is
introduced into a-site
- this is equilibrium reaction
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Chain Elongation – Transpeptidation
Step 2
- peptidyl-transferase ribozyme (23S) RNA molecule in small subunit of
prokaryotic ribosome
othis is catalyzing substance
oMOST IMPORTANT IN CATALYSING PEPTIDE BOND FORMATION
oForms peptide bound between p site and a site
oWhole large subunit then rachets to next codon
- Bond between P-site aa and tRNA is transferred to
amino group of A-site amino acid
oPeptide bond formed
otRNA is displaced
ono cofactors (high energy bond)
- small subunit shift with respect to large subunit
Stage 3: Translocation
- small subunit moves 3 bases 5’ 3’ on mRNA
- ribosome returned to original conformation
ouncharged P-site tRNA
oe site is expelled
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