Translation 3- Initiation

Question 1

What are ribosomes

Answer 1

1. Molecular machines that catalyze peptide bond formation directed by information in mRNA

Question 2

What does polycistronic mean and give example

Answer 2

1. Multiple genes encoded on a single messenger
2. Bacterial mRNA

Question 3

What is approx speed of translation, transcription and replication in bacteria and eukaryotes

Answer 3

1.Translation: add 15 amino acids per sec.
2. Transcription: add about 50 nucleotides per sec
3. Replication is much faster: 1000 nucleotides per sec
4. Euk- translation elongation is about 2 amino acids per sec

Question 4

Describe the general composition of ribosomes

Answer 4

1. Eukaryotes are bigger
2. All have large and small subunit consisting of proteins and RNA
3. Mostly made up of rRNA
4. 60% RNA and 40% protein

Question 5

Describe prokaryotic ribosome structure

Answer 5

1. 70S = size of prokaryotic ribosome
2. Large subunit = 50S
3. Small subunit = 30S
4. Doesn’t add up to 70S as surface area increases as you split them up

Question 6

Describe composition of small ribosomal subunit in bacteria

Answer 6

1. 16S rRNA (bacteria)
   2 .21 proteins (bacteria)

Question 7

Describe composition of small ribosomal subunit in eukaryotes

Answer 7

1. 18S rRNA (eukaryotes)
2. 33 proteins (eukaryotes)

Question 8

What is main role of small ribosomal subunit

Answer 8

1. Initial binding of mRNA and initiator met-tRNA

Question 9

Describe composition of large ribosomal subunit in bacteria

Answer 9

1. 23S & 5S rRNA (bacteria)
2. 31 proteins (bacteria)

Question 10

Describe composition of large ribosomal subunit in eukaryotes

Answer 10

1. 28S, 5.8S, & 5S rRNA (eukaryotes)
2. 49 proteins (eukaryotes)

Question 11

What are the 3 steps of translation cycle

Answer 11

1. Initiation – small unit binding to initiator tRNA
2. Elongation
3. Termination and recycling

Question 12

What direction does polypeptide synthesis occur

Answer 12

1. From N-terminus to C-terminus

Question 13

What direction do ribosomes read mRNA

Answer 13

1. From 5’ to 3’ direction
2. Accounts for observation that ribosomes initiate translation on nascent mRNAs

Question 14

How does chain elongation occur

Answer 14

1. By the linkage of the growing polypeptide to the incoming tRNA’s amino acid residue
2. Amino acid residues are sequentially added to the C-terminus of the nascent, ribosomally bound polypeptide chain
3. Nascent polypeptide must grow by being transferred from one peptidyl-tRNA to the incoming aminoacyl-tRNA to form a peptidyl-tRNA with one more residue

Question 15

What are the three tRNA-binding site on the ribosome

Answer 15

1. Peptidyl- p-site- binds the peptidyl-tRNA
2. aminoacyl or A-site - binds the incoming aminoacyl-tRNA
3. Exit site - binds outgoing deacylated tRNA

Question 16

What is the initiator codon and how is its position mediated in E.coli

Answer 16

1. AUG
2. Mediated by base pairing between the ribosome-binding site in the 5’ untranslated region and the 3’ end of the 16s rRNA
3. 16S Rrna determines where the AUG codon sits

Question 17

.What are the Factors and components needed for initiation

Answer 17

1. Small ribosomal subunit
2. mRNA
3. Initiation factors – proteins that are not part of the ribosome- soluble proteins
4. fmet-tRNAfMet – initiator tRNA
5. GTP – hydrolysis, initiator factor 2 requires GTP
6. Large subunit

Question 18

Describe structure of mRNA

Answer 18

1. C rich region – 16S rRNA – positions messenger where it needs to be
2. Base pairing to RBS – Ribosomal binding site- G rich - Shine-Dalgarno sequence

Question 19

What is fMET

Answer 19

1. fMet is the N-terminal residue of prokaryotic polypeptides
2. has been formylated- It has already an amide bond so can only be the N-terminal residue of polypeptide
3. Resembles peptide bond- mimics
4. An example of mimicry in translation

Question 20

How is the translational initiation site selected

Answer 20

1. The masking of AUGs that are not initiation codons by mRNA secondary structure
2. Interactions between the mRNA and the 16S rRNA that select the initiating AUG

Question 21

What are the initiation factors involved in initiation

Answer 21

1. IF-1
2. IF-2
3. IF-3

Question 22

What are translation factors

Answer 22

1. proteins used at only one step of the translation process
2. Not permanent components of the ribosome, but cycle on and off

Question 23

What does IF-1 do

Answer 23

1. Assists binding of IF-3 to the 30S ribosomal subunit.
2. IF-1 also occludes the A site (blocks) of the small ribosomal subunit, helping ensure that the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation.

Question 24

What does IF-2 do

Answer 24

1. IF-2 is a small GTP-binding protein.
2. IF-2-GTP binds the initiator fMet-tRNAfMet & helps it to dock with the small ribosome subunit.

Question 25

What does IF-3 do

Answer 25

1. IF-3 binds to the 30S ribosomal subunit, freeing it from its complex with the 50S subunit. 2. Breaks up small and large subunit

Question 26

Describe the first step of initiation

Answer 26

1. On completing a cycle of polypeptide synthesis the 30S and 50S subunits are separated 2. IF-3 then binds to the 30S subunit to prevent the reassociation of the 50S subunit 3. IF-3 appears to bind to upper end- doesn't function by physically blocking the binding of the 50S subunit

Question 27

Describe the second step of initiation

Answer 27

1. mRNA and IF-2 in a ternary complex with GTP and fMet-tRNAfMet accompanied by IF-1 subsequently bind to the 30S subunit in either order 2. Recognition not mediated by codon-anticodon interaction 3. Interaction helps bind fMet-tRNAfMet to the ribosome 4. IF-1 binds in the A site where it may function to prevent the inappropriate or premature binding of a tRNA 5. IF-3 destabilises the binding of tRNAs that lack the 3 G-C pairs in the anticodon stem of RNAfMet and helps discriminate between matched and mismatched codon-anticodon interactions

Question 28

What is the 3rd step of initiation

Answer 28

1. 50S subunit joins the 30S initiation complex in a manner that stimulates IF-2 to hydrolyse its bound GTP to GDP+P 2. This irreversible reaction conformationally rearranges the 30S subunit and releases IF-2 for participation in further initiation reactions

Question 29

What does initiation result in

Answer 29

1. In formation of an fMet-tRNAfMet -mRNA-Ribosome complex in which the fMet-tRNAfMet occupies the P-site while its A site is poised to accept an incoming aminoacyl-tRNA

Question 30

How is fMet-tRNAfMet formed

Answer 30

1. In E.coli uncharged (deacylated) tRNAfMet is first aminoacylated with methionine by the same MetRS that charges the tRNAmMet 2. Resulting Met-tRNAfMet is specifically N-formylated to yield fMet-tRNAfMet

Question 31

What is difference between tRNAfMet and tRNAmet

Answer 31

1. Have same anticodon sequence- recognised by same synthetase 2. Lack of base pair for fMet- important for formylation 3. Base pair means tRNAmet is not recognised by transformylation