Protein Synthesis - Basic Principles I

Question 1

Protein synthesis is ?

Answer 1

The conversion of genetic code into enzymatic and structural components of a cell

Question 2

What about the types of RNA involved in translation?

Answer 2

1. mRNA
   - Genetic information (sets of three nucleotides known as codons)
2. tRNA
   - Deciphers the genetic code (triplet codons). The adaptor between the genetic code and the amino acids
   - Each type is specific for one type of amino acids (20 amino acids)
   - Deliver the amino acid to the correct location on the ribosome
3. Ribosomal RNA (rRNA)
   - Part of the ribosome - Interact with ribosomal proteins, mRNA and tRNA

Question 3

What does ribosome recognise during initiation in Eukaryotes ?

Answer 3

Ribosome recognises 5’CAP

Question 4

Where does Prokaryotic translation occur?

Answer 4

• Cytoplasm
• Multiple ribosome can engage with each message even before it is fully transcribed
• Message can be polycistronic (more then one coding region)

Question 5

Where does translation occur in Eukaryotes?

Answer 5

• Message made in the nucleus

- Translation in the cytoplasm or on the surface of the ER

Question 6

How many types of tRNA are in Prokaryotes and Animals&Plants ?

Answer 6

• Prokaryotes 30-40 types

- Animals and Plants 50-100

Question 7

As tRNA greatly exceeds the number of amino acids used in protein synthesis (20). What does this result to for the amino acids ?

Answer 7

Thus, amino acids can have more then one tRNA

Question 8

What codon is ATG (AUG) ?

Answer 8

Start codon

- codes for formyl-methionine (prokaryotes) and methionine (eukaryotes)

Question 9

What codon is UAA, UAG, UGA ?

Answer 9

Stop codons

Question 10

Where are anti-codons found?

Answer 10

tRNA

Question 11

Why does Wobble base pairing occur ?

Answer 11

The Wobble Hypothesis explains why multiple codons can code for a single amino acid. One tRNA molecule (with one amino acid attached) can recognise and bind to more than one codon, due to the less-precise base pairs that can arise between the 3rd base of the codon and the base at the 1st position on the anticodon

Question 12

Ribosome are made up of two subunits. The size of ribosome and their subunits are measured in ?

Answer 12

Sedimentation velocity (Svedberg units)

Question 13

What is Translation ?

Answer 13

A step in protein biosynthesis where the genetic code from a strand of mRNA is decoded to produce a particular sequence of amino acids

Question 14

What are the steps of Translation ?

Answer 14

1. Bio-activation (attachment of correct amino acid to tRNA)
2. Initiation (Binding to the initiator codon = AUG)
3. Elongation (extension of the polypeptide chain)
4. Termination (release of the nascent polypeptide signalled by a stop codon)

Question 15

Give an overview of Bioactivation (Step 1) ?

Answer 15

The linking of an amino acid to its tRNA

• Catalysed by an aminoacyl-tRNA synthetase
• Each aminoacyl-tRNA synthetase is specific for an amino acid
• Thus there are 20 aminoacyl-tRNA synthetases
• Each being specific for all the tRNA’s for a particular amino acid (recognise the anti codon loop)

Question 16

Explain Bio-activation - charging the tRNA ?

Answer 16

• An ATP dependent process
• AMP is attached to the amino acid (releases diphosphate)
• Anti-codon loop provides specificity for the tRNA binding
• The amino acid is attached to the 2’ or 3’ hydroxyl of the terminal adenine of tRNA, Creating a high energy bond and releasing AMP
  Nucleophilic reaction
• The energy of the ester bond between the amino acid and the AMP drives the formation of charged tRNA

Question 17

What are the main features of the ribosome ?

Answer 17

• E (exit) site
• P (peptidyl) site
• A (aminoacyl) site
• Polypeptide exit tunnel
• mRNA-binding site

Question 18

What happens during Prokaryotic Initiation ?

Answer 18

• Separation of the ribosome units

- Binding IF1 and IF3 separate the subunits of the ribosome

Question 19

What do IF1 and IF3 do ?

Answer 19

IF1 - blocks the A site

IF3 - prevents large ribosome subunit binding

Question 20

What does IF1-30S-IF3 complex allow ?

Answer 20

IF1-30S-IF3 complex allows binding of the 30S ribosome component to mRNA

Question 21

What binds the shine Dalgarno site ?

Answer 21

16s rRNA

Question 22

What does IF2 deliver ?

Answer 22

IF2 delivers the formyl-methionine to the start codon

Question 23

What happens during completion of Initiation ?

Answer 23

• IF2-GTP-fMet-tRNA promotes association of the 50S subunit

- Correct positioning of the fMet tRNA causes IF2-GTP to be hydrolysed and IF2 is ejected

Question 24

What happens during Eukaryotic Initiation ?

Answer 24

• The 43S preinitiation complex

- 43S preinitiation complex is formed by a number of eIFs and Met-tRNA

Question 25

Formation of the 48S pre-initiation complex ?

Answer 25

Scanning mediated by the kozac AUG sequenceand base pairing occurs

Question 26

Explain Formation of the 48S pre-initiation complex ?

Answer 26

60S subunit delivered by eIF5B-GTP binding to eIF1A
- Binding of 60S is mediated by eIF5B which delivers the large subunit (binds to eIF1A) - eIF2 hydrolyses GTP and all initiation factors ejected, except eIF1A/5B

Question 27

Explain Large 60S subunit binding ?

Answer 27

• Correct association of ribosome units leads to GTP hydrolysis by eIF5B and release of all remaining factors
• The poly A tail complex can now re-associate with the 5’ CAP complex