5. protein synthesis Flashcards
vital functions of ribosome
- bind codons that permits high fidelity reading of codons
- includes binding sites for tRNA molecules
- interact with protein factors required for polypeptide chain initiation, elongation and termination
- catalyse peptide bond formation
- undergo movement permitting translational sequential codons
sites of ribosomes
A site (aminoacyl): accepting aminoacyl tRNA P site (peptidyl): accomodates the tRNA attached to growing peptide chain E site (exit): leaving of tRNA without aa
A and P site tRNAs interact closely together for peptidyl transferase reaction
how does a tRNA bind to ribosome
tRNA binds to 30s (small subunit)- anticodon arm and the rest of of tRNA bound to 50s
RNA to RNA contact (targets universally conserved regions)
allow for peptide bond to form between amino acids
what happens as the polypeptide extends
fits into tunnel on 50s subunit that extends from P site to the outer ribosomal surface
how are eukaryotic ribosomes different
3tRNA binding sites- different- larger 80s (40s, 60s)
Why eukaryotic ribosomes different
have to interact with nuclear membrane and secretory system (golgi and ER)
how do ribosomes operate
- synthesis proceeds from N to C terminus (5’ to 3’)
2. new peptidyl-tRNA is transferred from A to P- uncharged tRNA moves to E site
why can’t eurkaryotic translation begin immediately
mRNA must be translocated out of nuclear membrane
polysome
more than one ribosome is translating on mRNA at the same time due to length of mRNA
Translation initiation:
assembly of translation system components before peptide formation
Translation initiation: requires
initiator tRNA and initiation factors. in Ecoli initiator is N-formulated Met residue (AUG)
how does e.coli ribosome know where to start from
Shine-Dalgarno sequence upstream to start codon of mRNA
16s of 30s rRNA contains special sequence after start codon
mRNA contains complementary region (SD sequence)
Translation initiation: initiation factors
IF 1,2,3
how do eukaryotes know where to start translation from
cap that ribosomes bind and leave on 40s
what does initiation result in
fMet- tRNAfmet x mRNA x ribosome complex
with start codon under P site
IF2 GTP brings charged tRNA to P site
first tRNA binds with fMet IF2 hydrolysis of GTP
allows second half of ribosome to be clipped on (50S attaches forming 70S subunit)
tRNAfmet is the only tRNA that enters P site
all other enter A site during chain elongation
what does eukaryotic imitation require
ATP cap at 5’ mRNA
recognise mRNA’s initiating AUG codon embedded in consensus sequence
7-methylguanosine cap and are monocistronic
translation elongation stages
decoding
transpeptidation
translocation
decoding
ribosomes select and binds an aminoacyl-tRNA of complementary anticodon to codon on mRNA on A site
transpeptidation
peptide bond formation
peptidyl group in P site tRNA transferred to aminoacyl group in A site
translocation
a and p site tRNAs transferred to P and E sites
uncharged tRNA from P to E
peptidyl tRNA from A to P
delivery of next aminoacyl tRNA by elongation factors
requires GTP hydrolysis bring into A site
peptide bond formation enzyme
peptidyl transferase in large subunit
termination codon
no tRNA bound
UAA UGA UAG
recognised by release factors 1 and 2 GTP hydrolysed
proteins and sub units break off- recycled
decoding detail
ribosomes monitor correct codon-anticodon pairing
EF-Tu x GTP delivers aa-tRNA to ribosome
tRNA’s aminoacyl end moves into A site after GTP os hydrolysed and EF-Tu dissociates
transpeptidation detail
ribosome is a riboenzyme
peptidyl transfer centre on large subunit- consists of rRNA
nearest protein side chain is 18 aa away from newly formed peptide bond
nearest Mg ion is 8.5 aa away
Puromycin
binds to A site without need for elongation factors
streptomycin
member of aminoglycosides
ribosome misreads mRNA binds to 30S
Chloramphenicol
inhibit peptidyl transferase activity
tetracycline
binds to 30s
prevents entry to aminoacyl tRNA into A site but allows Ef-Tu to hydrolyse GTP- energetic drain on cell
Ricin
removes highly consvered adenine base of 28S stops ribosomes binding
how do you fold a protein
ribosome associated chaperones
why do you need chaperones
ribosomes exit channel is too narrow to allow secondary structure formation
unfolded hit with other proteins causing misfolding
Trigger factor
recruited by ribosomal proteins recognise short hydrophobic protein segments does not require ATP prevents mis folding and aggregation near exit of ribosomes tunnel
Proteolysis
activate proproteins- cleavage
contain signal peptide to be excised
zymogen
inactive enzymes
ubiquination
sends protein for degradation
glycosylation
to signal transfer of protein to lumen of ER from cytosol
