Flashcards in Protein Metabolism I Deck (36)
responsible for attaching amino acids to the 3' CCA end of a tRNA
UAA, UAG, UGA
Canonical base pairing w/1st two codon bases (normal base rules); non canonical base pairing is possible at third site (non-normal base pairing);
Why is the wobble effect hypothesis important?
1) kinetic advantage - faster protein synthesis 2) tRNA can dissociate more readily from the RNA template.
How do aminoacyl-tRNA synthetases apply the correct amino acid?
They check for base pairing within' the tRNA, and they also analyze modified bases.
Aminoacyl tRNA synthetase Reaction
Goal: form an ester linkage between AA and tRNA. 1) charged with ATP forming AA-AMP intermediate. 2) Aminoacyl-AMP + tRNA goes to Aminoacyl-tRNA + AMP
Class I aminoacyl-tRNA synthetases
1) Accommodate larger, more hydrophobic amino acid substrates; 2) enzymes are monomers
Class II aminoacyl-tRNA synthetases
1) Accommodate smaller, more hydrophillic amino acid substrates 2) enzymes are dimers or multimers.
Step 2 difference between class I and class II aminoacyl-tRNA synthetases
1) class I aminoacyl-tRNA synthetases transfer AA to the 2'OH first then to the 3' OH via a transesterification reaction. 2) class II transfers AA directly to 3'OH
Aminoacyl-tRNA synthetase sites
site 1: catalytic domain (interacts with the tRNA 3'end to load AA) ; site 2: variable domain ( interacts with the specific bases on the tRNA that identify the tRNA)
Specificity of aminoacyl-tRNA synthetases to tRNA
1) one or more bases in anticodon 2) one or more bases in the acceptor stem 3) discriminator base 73
Where does high fidelity of tRNA synthetases come from?
1) Size exclusion 2) editing pre-transfer 3) editing post-transfer.
aminoacyl-tRNA synthetase accomodates activated amino acid; if rejected pre-transfer, then aminoacyl-adenylate is hydrolyzed
amino-acyl tRNA synthetase; if rejection occurs post transfer AA is cleaved from tRNA
helicase; uses atp; pulls mRNA transcripts out of the nuclear pore
Because prokaryotic mRNA is not capped, how are they recognized by ribosomes?
1) 3' end of 16srRNA is recognized by purine rich shine-dalgarno sequence
Initiation of transcription in Eukaryotes
1) Ribosome binds to 5' cap with the aid of proteins IRES 2) Ribosome scans for AUG
1) DNA segment that leads to the production of 30S subunit, 50S subunit; and the tRNA in prokaryotes. 2) methylation of pre-rRNA transcript 3) methylation guides proteins to cut transcript.
antibiotic; binds to the ribosomal catalytic site blocking protein synthesis.
GTPase- activating protein; promotes the hydrolysis pf GTP by EFTu once aminoacyl-tRNA is in the A site
guanine-nucleotide exchange factors; recharge GDP to GTP
1) Ribosome recognizes shine-dalgarno sequence. 3' end of the ribosome has a pyrimidine rich sequence that recognizes purine rich segment on 5' end of mRNA.
30S initiation complex components
1) 3x IF-x 2) GTP 3) fmet-tRNAfmet, mRNA, and 30S ribosomal subunit.
elongation factor that brings tRNA to ribosome; formal group inhibits fmet-tRNAmet from interacting with EFTu; formal groups only in prokaryotes.
What two regions distinguish initiator met tRNA from other tRNAs?
3x GC region; C_A (non-bP) region.
initiation factor 3; binds to E site of the ribosome; prevents aminoacyl-tRNA from binding to E site and prevents 50s from binding to the 30s
binds 30S subunit in A site. prevents tRNA from binding to the A site
Pre-initiation complex formation Prokaryotes
1) IF-3 binds the E site of the 30S subunit, IF-1 binds the A site, mRNA is recruited with the help of the 16s RNA (recognizes the shine delgarno sequence) 2) IF-2-GTP binds 30S and recruits fmet-tRNAmet
70 Initiation complex
1) As soon as the initiator tRNA arrives in the P site, IF-3 leaves, which allows the 50S to bind to the 30S 2) 50S acts as a GAP hydrolyzing IF-2-GTP causing IF-2 and IF-1 to leave.
Eukaryotic 40S initiation complex/transcription initiation overview.
1) EIF-1 binds E site; EIF1A binds to A site; EIF3 binds to the backside of the 40S subunit 2) EIF4F preps mRNA; EIF-2 binds to tRNA and guides it to P site along with EIFB-GTP and EIF-1A (In this case, the initiator tRNA is not guided by the mRNA transcript. 3) EIF4F complex aids in the loading with the entire ribosomal complex. 4)codon-anticodon interaction causes GTP hydrolysis of EIF2`
Complex that binds to mRNA before loading onto mRNA. has a segment that binds to the 5' cap (EIF4E), the polyA tail (PABP) another just makes contact, and one has ATPase activity. ATPase activity helps complex weave through mRNA until it finds start codon AUG for anticodon-codon interation.
PolyA binding protein
(PAPB) Binds polyA tail and EIF4 to stabilize the ribosome mRNA interaction for translation.
Elongation Process in prokaryotes
1) EF-Tu binds aminoacyl-tRNA and GTP 2) complex binds to A site, GTP is hydrolyzed and EF-Tu GDP complex dissociates. 3) 23S subunit forms catalytic site. Nucleophillic attack of AA in A site to AA in P site 4) EF-G-GTP causes translocation of tRNA in A site to the P site.
Protein that binds to the tRNA in A site causes activation of GTPase ability, pushes tRNA in A site into the P site.
Bind to interaction with stop codons; causes hydrolysis of peptide chain.