Mechanism & regulation of translation I

Question 1

what is the central dogma?

Answer 1

theory stating that genetic information flows only in one direction, from DNA to RNA, to protein or RNA directly to protein

Question 2

protein synthesis

Answer 2

• crucial for cell growth, proliferation and survival
• expensive process for the cell, therefore tightly regulated

Question 3

what can protein synthesis regulation do?

Answer 3

control overall rates of proteinsynthesis and modulate the expression of specific transcripts

Question 4

what can proteinsynthesis be inhibited by?

Answer 4

cell stresses and withdrawal of nutrients; serum deprivation, temperature shock, DNA damage, viral infection, hypoxia, cytokine treatment

Question 5

prokaryotes

Answer 5

• polycistronic
• normally unstable
• translation can occur on nascent transcripts

Question 6

eurkaryotes

Answer 6

• normally monocistronic
• capped and polyadenylated
• 5’ and 3’ UTR

Question 7

what is the structure of CAP?

Answer 7

• found at the 5’ end of all cellular mRNAs
• seals end of mRNA protecting it from nuclease digestion
• landing pad for elF4E

Question 8

what is the process of capping of newly synthesised mRNAs?

Answer 8

1. removal of 5’ terminal phosphate (triphosphatase)
2. addition of 5’ terminal GMP (guanylyl transferase)
3. methylation of guanine base (guanine- 7methyl transferase)
4. methylation of ribose (in some cases)

Question 9

what is polyadenylation?

Answer 9

• 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

Question 10

what are the stages in the 3’ polyadenylation of newly synthesised mRNAs?

Answer 10

• 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

Question 11

what are the general characteristics of tRNA molecules?

Answer 11

• 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

Question 12

ribosome structure

Answer 12

large and small subunits
contain 50% proteinsand 50% rRNAs (by mass)

Question 13

function of 80s ribosomes

Answer 13

• 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

Question 14

what are the three binding sites for tRNA in ribosomes?

Answer 14

E; exit site
P; peptidyl-RNA binding site
A; aminoacyl-tRNA binding site

Question 15

other facts

Answer 15

• 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

Question 16

CAP-dependent protein synthesis; initiation

Answer 16

• small ribosomal subunit and initiatior tRNA bind, recognition of CAP, scanning to start AUG codon
• ternanry complex binds large ribosomal unit

Question 17

CAP-dependent proteinsynthesis; elongation

Answer 17

• 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)

Question 18

CAP-dependent proteinsynthesis; termination

Answer 18

• at 3’ end of coding sequence the ribosome encounters a stop codon
• polypeptide chain released

Question 19

initiation process

Answer 19

• 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

Question 20

three main steps of initiation

Answer 20

1. assembly of 43s pre-initiation complex
2. binding of mRNA to 43s complex
3. assembly of 80s initiation complex

Question 21

what is the structure of polypeptide chain eIF2

Answer 21

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

Question 22

1. assembly of 43S pre-initiation complex

Answer 22

association met-RNA with eIF2/GTP (ternary complex)
40S trapped as monomer by eIF2 (43S)
binding of complexes to form 43S pre-initiation complex

Question 23

2. binding of mRNA to 43S complex

Answer 23

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

Question 24

role of eIF4E

Answer 24

recognition of 5’ CAP on mRNA

Question 25

role of eIF4G

Answer 25

binds to eIF4E, eIF4A and eIF3

Question 26

role of eIF3

Answer 26

acts as a bridge between eIF4G and the 40s ribosomal subunit, hence required for mRNAs binding to the ribosome

Question 27

role of eIF4A

Answer 27

helps unwind secondary structure in 5' end of the mRNA

Question 28

structure of eIF4G

Answer 28

- has multiple domains for interaction with other initiation factors - acts as scaffold molecule that brings together other translation IFs

Question 29

interaction of 43S pre-initiation complex with 3' end of the mRNA via poly(A) tail

Answer 29

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

Question 30

what happens after binding of 43S pre-in. comlex to CAP site?

Answer 30

scanning to the initiating AUG

Question 31

what is the effect of secondary structure in the 5' UTR of an mRNA?

Answer 31

impedes the attachment of the 43S pre-initiation complex to the mRNA and/or reduces ability of the complex to scan along the mRNA

Question 32

IRES; internal ribosomal entry site

Answer 32

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

Question 33

3. assembly of the 80S initiation complex

Answer 33

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

Question 34

what does assembly of the 80S initiation complex involves?

Answer 34

- 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)

Question 35

stages in the formation of the first peptide bond and elongation

Answer 35

- AA-tRNA binding; catalysed by eIF1, requires GTP - peptide bond formation; catalysed by the ribosome itself - translocation; catalysed by eEF2, requires GTP

Question 36

how is STOP codon recognised?

Answer 36

by release factor eRF STOP codons; - UAA ochre - UAG amber - UGA opal

Question 37

what happens at and after peptide formation?

Answer 37

- hydrolysis of the last peptidyl-tRNA bond and release of the completed polypeptide chain - dissociation of the two ribosomal subunits from the mRNA

Question 38

what is a polysome?

Answer 38

structure that consists of multiple ribosomes attached to a single mRNA

Question 39

free ribosomes

Answer 39

synthesise soluble proteins that function in the cytosol

Question 40

ER bound ribosomes

Answer 40

synthesise proteins destined either for incorporation into cell membranes or for export from the cell