Mechanism & regulation of translation I Flashcards
what is the central dogma?
theory stating that genetic information flows only in one direction, from DNA to RNA, to protein or RNA directly to protein
protein synthesis
- crucial for cell growth, proliferation and survival
- expensive process for the cell, therefore tightly regulated
what can protein synthesis regulation do?
control overall rates of proteinsynthesis and modulate the expression of specific transcripts
what can proteinsynthesis be inhibited by?
cell stresses and withdrawal of nutrients; serum deprivation, temperature shock, DNA damage, viral infection, hypoxia, cytokine treatment
prokaryotes
- polycistronic
- normally unstable
- translation can occur on nascent transcripts
eurkaryotes
- normally monocistronic
- capped and polyadenylated
- 5’ and 3’ UTR
what is the structure of CAP?
- found at the 5’ end of all cellular mRNAs
- seals end of mRNA protecting it from nuclease digestion
- landing pad for elF4E
what is the process of capping of newly synthesised mRNAs?
- removal of 5’ terminal phosphate (triphosphatase)
- addition of 5’ terminal GMP (guanylyl transferase)
- methylation of guanine base (guanine- 7methyl transferase)
- methylation of ribose (in some cases)
what is polyadenylation?
- poly A tail found at 3’ end of mRNAs
- protects mRNA from enzymatic degradation
- aids in transcription termination, export of mRNA from the nucleus and translation
what are the stages in the 3’ polyadenylation of newly synthesised mRNAs?
- recognition of AUAAA sequences by specificity componens RNA cleavage by cleavage factors
- initial poly(A) polymerisation by poly(A) polymerase; followed by binding of poly(A) binding protein (PABP)
- more poly(A) polymerisation and binding of more PABP
what are the general characteristics of tRNA molecules?
- single RNA strand of approx 80 nucleotides
- helps decode mRNA sequence into a protein
- function at specific sites in the ribosome during translation
- aminoacyl tRNA synthestase links amino acid to 3’ end of acceptor arm to produce an aminoacyl-tRNA
ribosome structure
large and small subunits
contain 50% proteinsand 50% rRNAs (by mass)
function of 80s ribosomes
- small and large subunits bind together during initiation
- translation takes place in the cavity between the two subunits
- peptidyl transferase activity associated with the large 60s subunit
what are the three binding sites for tRNA in ribosomes?
E; exit site
P; peptidyl-RNA binding site
A; aminoacyl-tRNA binding site
other facts
- translation must go fast enough to supply protein but slow enough to avoid too many errors
- error rate; 1 in 10^4 incorrect aa
- ribosomes add 20 aa/second to a polypeptide chain
- proteinsynthesis is energetically expensive
CAP-dependent protein synthesis; initiation
- small ribosomal subunit and initiatior tRNA bind, recognition of CAP, scanning to start AUG codon
- ternanry complex binds large ribosomal unit
CAP-dependent proteinsynthesis; elongation
- tRNA brings aa to the ribosome in order specified by codons on mRNA
- ribosome catalyses peptide bond formation between amino group of each aa
- more than one ribosome is active on any one mRNA (polysome)
CAP-dependent proteinsynthesis; termination
- at 3’ end of coding sequence the ribosome encounters a stop codon
- polypeptide chain released
initiation process
- rate limiting step and tightly regulated
- > 10 soluble eukaryotic initiation factors (eIF)
- aim is to bring mRNA to ribosome and initiator tRNA to AUG start codon
three main steps of initiation
- assembly of 43s pre-initiation complex
- binding of mRNA to 43s complex
- assembly of 80s initiation complex
what is the structure of polypeptide chain eIF2
three subunits;
- GTP binding site; on y subunit
- phosphorylation site; found on a subunit, ser 51
- K boxes on b subunit, involved in interaction of eIF2B and eIF5
- assembly of 43S pre-initiation complex
association met-RNA with eIF2/GTP (ternary complex)
40S trapped as monomer by eIF2 (43S)
binding of complexes to form 43S pre-initiation complex
- binding of mRNA to 43S complex
eIF4E binds to CAP on mRNA
eIF4G binds to EIF4E
eIF4A unwinds mRNA secondary structure
43S preinitiation complex binds to eIF4G (via eIF3)- 48S preinitiation complex
scanning
role of eIF4E
recognition of 5’ CAP on mRNA
role of eIF4G
binds to eIF4E, eIF4A and eIF3
role of eIF3
acts as a bridge between eIF4G and the 40s ribosomal subunit, hence required for mRNAs binding to the ribosome
role of eIF4A
helps unwind secondary structure in 5’ end of the mRNA
structure of eIF4G
- has multiple domains for interaction with other initiation factors
- acts as scaffold molecule that brings together other translation IFs
interaction of 43S pre-initiation complex with 3’ end of the mRNA via poly(A) tail
interaction achieved by ability of poly(A) binding protein (PABP) to bind to both eIF4G and eIF4B that is part of 43S pre-in. complex
= circulatisation of the mRNA by assocation of the 5’ end with the 3’ end
what happens after binding of 43S pre-in. comlex to CAP site?
scanning to the initiating AUG
what is the effect of secondary structure in the 5’ UTR of an mRNA?
impedes the attachment of the 43S pre-initiation complex to the mRNA and/or reduces ability of the complex to scan along the mRNA
IRES; internal ribosomal entry site
allow direct binding of the 43S complex to the mRNA without the need or CAP recognition or scanning
- IRES can replace function of some of the IFs
- some viral mRNA e.g. picornacirus use IRES mediated CAP-independent translation
- assembly of the 80S initiation complex
40S/43S preinitiation complex scans to the AUG start codon
Met tRNA occupies the P site on the ribosome
60S subunit associated to form the 80S complex
what does assembly of the 80S initiation complex involves?
- binding of the 60S ribosomal subinit to the 43S mRNA complex
- hydrolysis of the GTP bound to eIF2
- release of most of the IFs from the ribosome
- these events need eIF5 (has GTPase activity)
stages in the formation of the first peptide bond and elongation
- AA-tRNA binding; catalysed by eIF1, requires GTP
- peptide bond formation; catalysed by the ribosome itself
- translocation; catalysed by eEF2, requires GTP
how is STOP codon recognised?
by release factor eRF
STOP codons;
- UAA ochre
- UAG amber
- UGA opal
what happens at and after peptide formation?
- hydrolysis of the last peptidyl-tRNA bond and release of the completed polypeptide chain
- dissociation of the two ribosomal subunits from the mRNA
what is a polysome?
structure that consists of multiple ribosomes attached to a single mRNA
free ribosomes
synthesise soluble proteins that function in the cytosol
ER bound ribosomes
synthesise proteins destined either for incorporation into cell membranes or for export from the cell
