Ch273- Ch22
tRNA binding at A site
--> CC exposed ester bond in P site--> P site becomes deacylated--> moves to E site. peptidyl tRNA moves to P site. mRNA advances through the ribosome by one codon. the formation of a peptide bond causes the ribosome to loosen its grip on the mRNA
GtP hydrolysis in termination
--> dissociation of RF's--> ribosome has bound mRNA, empty A site, and deacylated tRNA in the P site. RRF slips into A site--> EFG binding translocated RRF to P site--> displacement of deacylated tRNA.
wobble hypothesis
3rd codon position and 5' anticodon position experience flexibility (wobble) in H bonding geometry. you can bind more codons with less variable tRNA's. allows nonstandard AA's to be incorporated in stop codon positions.
3 sites
A site- accommodates incoming aminoacyl tRNA P (peptidyl site)- binds the tRNA with growing polypeptide chain E (exit) site- transiently binds a deacylated tRNA after peptide bond formation.
proofreading in synthetases
AA binding site is tailored to geometric and electrostatic properties of an AA--> less likely to activate incorrect AA. enhances specificity of tRNA aminoacylation.
GTP hydrolysis
GTP hydrolysis--> CC--> additional steps of reaction sequence.
ribozyme
RNA catalyst; forms bc transferase active site is in a highly conserved region of the large subunit.
protein pocket
a highly conserved protein pocket accommodates the aminoacyl group. all bind with the same affinity. one loop will bind a bit tighter than the other and vice-versa.
A site
aminoacyl tRNA must recognize a complementary mRNA codon in the A site. competition makes this a rate-limiting step of protein synthesis . before the large subunit catalyzes peptide bond formation, it must make sure the correct aminoacyl tRNA is in place. When tRNA binds the small subunit A site, two residues flip out to form h bonds with mRNA codon as it pairs with the tRNA anticodon--> physically links rRNA bases with the codon and anticodon so they can detect a correct match--> CC--> GTP hydrolysis--> leaves tRNA with aminoacyl group to be added to the growing polypeptide chain.
AA activation without tRNA
anticodon recognition site and aminoacylation active site communicate to guarantee correct AA attachment to tRNA.
aminoacylation
attachment of AA to tRNA; catalyzed by aminoacyl-tRNA synthetase (AARS
Shine-Dalgarno sequence
base pairs with a complementary sequence at the 3' end of the 16S rRNA--> positioning initiation codon in the ribosome.
P site
catalyzes peptide bond formation. prior to the formation of the first peptide bond, peptidyl transferase activity for the large subunit catalyzes a transpeptidation reaction where free AA of aminoacyl tRNA in the A site attacks the ester bond that links peptidyl group by one AA at its C-term= polypeptide grows from N-->C using energy from the broken ester bond
translation is efficient in vivo
cells maximize rate of protein synthesis by using polysomes
class I v Class II enzymes
class I- attach an AA to the 2' -OH group of tRNA class II- attach AA to the 3' -OH group of tRNA
3 initiation factors
initiation requires 2 IF's (1, 2, 3). IF3- binds to the small subunit to promote large and small subunit dissociation. uses assistance from IF2, a GTP binding protein. IF1- blocks A site of small subunit, forcing initiator tRNA to the P site.
ribosome
job is protein synthesis; linking of mRNA and AA's attached to tRNA's so AAs can be covalently linked in a specific order. contains RNA and proteins with 80% of the cells' RNA contained in there.
subunits
large and small subunits containing rRNA molecules. multi-domain structure--> conformational flexibility of small subunit. large subunit is rigid and immobile.
release factors
mediate translation termination. peptidyl group exits ribosome through a tunnel in the large subunit, which can shelter up to 30 residues; contains ribosomal proteins etc. tunnel surface is hydrophilic so no hydrophobic patches can impede the exit of an exiting polypeptide chain.
AARS
modular proteins with a catalytic domain where AA activation and transfer to a tRNA occur. must attach correct AA to tRNA bearing corresponding anticodon to ensure accurate translation. catalyzes formation of ester bond between AA and -OH group at 3' of tRNA--> aminoacyl tRNA. tRNA is now "charged" with an AA
translocation
movement of tRNA and mRNA that allows the next codon to be translated; requires G protein elongation factor G (efG)
where do tRNA and mRNA bind?
on a highly conserved rRNA-rich subunit interface.
initiation requires an initiator tRNA
protein synthesis begins at mRNA codon that specifies Met (AUG). in prokaryotes, the initiation codon lies 10 bases downstream a conserved mRNA sequence = Shine-Dalgarno sequence
initiator tRNA
recognizes the initiation codon charged with Met. doesn't recognize other Met sequences. in bacteria, Met attached to tRNA is modified by the transfer of a formyl group--> aminoacyl group fMet + initiator tRNA.
steps of aminoacylation
requires free energy of ATP. AA + tRNA + ATP--> aminoacyl tRNA + AMP + PPi 1) AA reacts with ATP--> aminoacyl-AMP. hydrolysis of PPi product makes this process irreversible 2) AA ("activated" by adenylation) reacts with tRNA--> aminoacyl tRNA and AMP.
How does rRNA catalyze peptide bond formation?
residues position the substrate for reaction = induced fit.
polysomes
single mRNA molecule simultaneously being translated by multiple ribosomes. after first ribosome clears start codon, second ribosome binds.
tRNA anticodons pair with mRNA codons
tRNA molecules align with mRNA codons, base pairing in an antiparallel fashion. many isoacceptor tRNA's can bind more than one of the codons that specify their AA.
elongation
the appropriate tRNA's are delivered to the ribosome during elongation. in each cycle, an aminoacyl tRNA enters the A site of the ribosome--> peptide bond formation--> tRNA moves to the P site, then to the E site. aminoacyl tRNA's are delivered to the ribosome in a complex with a GTP binding elongation factor (EF)
Q
transfer of peptidyl group from P site to water--> stable TS in hydrolysis reaction--> untethered polypeptide that can exit the ribosome.
when does translation stop?
when it reaches the stop codon. when stop codon is in A position, ribosome cant bind aminoacyl tRNA, but binds a release factor, which recognizes stop codons. simultaneously, a loop of RF with conserved sequence YYQ projects into peptidyl transferase site of large subunit
