biochem L23 + L24

translation of rna to protein

aminoacyl trna binds to ribosome one by one matching their anticodon to codon on mrna, the growing peptide chain is transferred from the first aminoacyl trna to the incoming aminoacyl trna, the first trna is released and ribosome move one codon length along the message allowing the next trna to come in, ribosomes encounters stop codon which releases the polypeptide chain

mrna

the template used to make a protein and read 5 to 3’

ribosome

uses mrna template and aminoacyl trna to make polypeptide

trna

transfers aa to the ribosome and delivers them and roping them off to an elongating peptide chain

aminoacyl trna formation

aminoacyl trna synthetase recognizes a particular aa and trna with corresponding anticodon and catalyzes the formation of aminoacyl trna with hydrolysis of one atp to amp

open reading frame

are mrna sequences bounded by start and stop codons that can be translated continuously

AUG codon

encodes n formylmethionine if used as start codon in prokaryotes and methionine if start codon in eukaryotes

what are 3 stop codons

uaa, uga, and uag and will release the polypeptide

organism codon bias

some organisms deviate from the standard genetic code encoding the 20 standard amino acids

modifications in codons

uga is a stop codon but it can code for selenocysteine

wobble hypothesis

the 5 prime base of anticodon can wobble in its position during translation which allows it to form alternative h bond arrangements with diff codon bases, g pairs with c or u, c pairs with g, a pairs with u, u pairs with a or g,

specificity in dna rep

how accurate the process is, proofreading by rna polymerase and h bonding btwn bases

efficiency

how quickly the process is done, done by open reading frames and polycistronic mrna

E coli lac operon

the mrna for this operon has open reading frames for lac z, lac y, and lac a genes each with start, stop, and shine dalgarno seq

shine dalgarno sequence

sequences help align mrna on the ribosome so translation begins at the right position

trna structure

it is a three loop structure held by h bonds, has acceptor stem loop, conserved anticodon loop at the bottom, unusual bases and base pairings

how do aminoacyl trna form

the amino acid is accepted by the synthetase and adenylated with aminoacyl adenylate bound to the enzyme, the proper trna is accepted by synthetase and aa is transferred to 3’ oh of 3’ terminal residue of trna or 2’oh followed by isomerization to 3’ aminoacyl trna

initiation factors in bacteria

if1 promotes dissociation of preexisting 70s and 80s, if2 helps attach initiator trna, if3 prepares mrna for ribosome binding

elongation factors in bacteria

ef tu helps deliver aminoacyl trna to ribosome, ef ts helps recharge ef tu with gtp, ef g helps facilitate translocation, ef p helps translation of consecutive proline codons

termination factors in bacteria

rf1 and rf2 are release factors, rf3 is gtpase that promotes release

ribosome percents

ribosome is made of 60 to 70 rna and 30 to 40 percent protein

bacterial ribosome

made of 50s and 30s to make 70s ribosome

eukaryote ribosome

made of 60s and 40s to make 80s ribosome

e coli 16s rrna structure

has three major domains of folding, highly conserved

crystal structures of 50s ribosomal subunit

shows peptidyl transfer site surrounded by rrna, peptidyl transfer site is far from protein indicating it is a ribozyme

crystal structure of 70 s ribosome

is in complex with mrna and two trnas, has peptidyl transferase, eftu complex region

Translation mechanism

initiation, elongation, termination, focus is on bacteria and archaea

translation Intiation process

if1 and if3 bind the 30s subunit, mrna binds 30s, if2GTP protein brings fmet trna to p site of 30s, 30s initiation complex forms with if1, if2, if3, and mrna, 50s joins the 30s and gtp hydrolyzed to gdp, ifs leave and initiator trna locked on p site to form 70s initiation complex

translation Elongation process

peptide chain is bound to trna at p site and e and a site is empty, the recognized aminoacyl trna binds in a site using eftu, eftu binds gtp which binds aminoacyl trna, eftu hydrolyzes gtp to gdp, peptide bond formed by peptidyl transferase, the peptide chain moves from trna in p site to a site to e site

translation termination process

ribosome finds stop codon with trna complementary to the codon, a release factor binds the a site, peptidyl trna is attacked by water which releases peptide, rf3 stimulates release process thru gtp binding and hydrolysis

Bacterial translation antibiotic inhibitors

streptomycin interferes with normal pairing btwn aminoacyl trnas and message codons, puromycin causes premature chain termination

eukaryotic translation inhibitor

cycloheximidine inhibits translation

translocation rate and coupling

translation in e coli is rapid which matches rate of transcription since rna polymerase and ribosome are on top of each other

bacterial protein secretion

new polypeptide chain complexes with sec b which prevents complete folding during transport to the membrane, at the membrane atpase sec a drives translocation thru the membrane with sec YEG to form membrane pore, the leader seq is cleaved off the secreted protein by membrane peptidase

in eukaryotes, proteins made in cytoplasm

unfolded protein attaches to tom complex, signal seq interacts with tim complex, electrochemical gradient across the inner membrane pulls the signal sequence thru, mitochondrial hsp 70 binds to the protein and pulls the rest of the protein thru, signal sequence is removed by mpp

membrane bound proteins made on rough er

signal seq is translated at 5’ end of mrna, recognition by srp further halts translation, srp recognizes docking protein on rer and signal seq is inserted into rer, protein synthesis is resumed, polypeptide chain is pulled thru the membrane and signal seq is cleaved off, protein in lumen of rer and ribosome is recycled

SNARES protein

vesicles with this protein interact and cause membrane fusion