6) Translation

Question 1

What is the overall process of translation?

Answer 1

1) Ribosome goes to mRNA to do the job of translation
2) tRNAs bring amino acids to the ribosome in an order specified by the mRNA sequence

RNA—–> Protein

Question 2

What are some of the components of translation?

Answer 2

Ones which will give special attention to:
Ribosomes
tRNA
Aminoacyl Synthetases

mRNA
Initiation Factors
Elongation Factors
Releasing Factors
Amino acids

Question 3

What is the structure of ribosomes?

Answer 3

Large and Small subunit

Both are made up of RNA and protein, with many different proteins per subunit

Overall secondary structure: RNA predominates

Large subunit: Made up of more than one RNA molecule= Bacteria: 23S and 5S, 3 in euk

Small subunit: Contain single RNA (bacteria= 16S and euk= 18S)

Question 4

What is the active site of a ribosome called?

Answer 4

Peptidyl Transferase
No ribosomal protein was near the active site
rRNA (ribosome RNAs)= between the 2 subunits, and is on the active site

Protein part of the ribosome is on the outward facing sides of the structure

Question 5

What do ribosomal RNAs do?

Answer 5

Fold up into complex secondary structures
rRNAS= Actually catalyse peptide bonds

rRNA is a single continuous molecule= has 4 separate domains

Important that a secondary structure is stable so that accessory proteins can attach to a scaffold

Can also have non-standard base pairs forming (e.g. G-U) in double stranded regions of an RNA molecule

Question 6

What do tRNAs do?

Answer 6

Bring amino acids to the active site of the ribosome

Amino acids are selected to be added to the growing polypeptide chain in an order specified by the sequence of the mRNA

Each amino acid is brought to the chain by transfer RNA specific for particular amino acid

Question 7

What are the 2 crucial regions of tRNA?

Answer 7

1) Anticodon= Complementary to the codon on mRNA
3’ end always ends in -CCA

2) Acceptor arm= Binds to amino acid
Formed by binding together of 7 bp of the 5’ and 3’ ends of the molecule, amino acid attaches to the adenosine at the 3’ end
Amino acid attaches at the adenosine at the 3’ end

Question 8

Where the specificity of tRNA come from?

Answer 8

Shape and modifications to tRNA

tRNA can then be recognised by its specific aminoacyl tRNA synthesise

Many different modifications= One of the most common is methylation of the base or sugar component of the nucleotide

Question 9

What does aminoacyl tRNA synthesase do?

Answer 9

Attaches an amino acid to tRNA
Key enzymes that power protein synthesis

Each one recognises the correct amino acid to link to the correct tRNA

One aminoacyl tRNA synthesise for each of the 20 amino acids

Aminoacyl= General term for amino acid

VERY specific + Activate amino acid with ATP
Energy maintained when linked to tRNA
Energy for protein synthesis comes from this chemical bond
Amino acid now passenger to the tRNA

Question 10

How does aminoacyl tRNA synthetase add amino acid to tRNA?

Answer 10

1) Activated amino acid intermediate has link between carboxyl group and AMP
ATP is cleaved= Produces energy to join amino acid to the tRNA
2) Amino acid-AMP remains bound to the enzyme and tRNA takes place of AMP= Forms aminoacyl-tRNA

Question 11

What are the 3 steps of translation? (Names only)

Answer 11

1) Initiation
2) Elongation
3) Termination
Just like transcription!

Question 12

What is the first step of translation?

Answer 12

1) Ribosomes: Large and small subunit remain separate in cytoplasm until need to assemble
2) Translation= Ribosome assembles on mRNA at ribosome binding site= Sequence upstream of first codon (AUG) that is complementary to short sequence of 16S rRNA
3) Initiator tRNA (Met-tRNAfmet ) is brought to the right position on the complex by IF-2 which accompanies the small subunit along with molecule of GTP= Energy while IF-3= Prevents large subunit from attaching until right time. This brings the Methionine amino acid
4) Large subunit attaches by using energy from GTP= Release of initiation factors= Makes process irreversible

Question 13

What are the different sites for binding tRNA molecules on ribosomes?

Answer 13

A= Aminoacyl site= tRNA must be bound to an amino acid that corresponds to the amino acid dictated by mRNA codon

P site= Peptidyl site= Holds tRNA bound to the growing polypeptide chain

E site= Exit= Releases now empty tRNA from the ribosome

Met-tRNAfMet is the only aminoacyl-tRNA that can load directly into the P-site. It does not need to go to the A site first.

GOES to A –> P —> E

Question 14

What is phase 2 of translation? (Overall)

Answer 14

ELONGATION

1) Individual amino acids are attached to the carboxyl (-COOH) terminus of the growing polypeptide= First peptide bond
2) Assembling the ribosome out of the 2 subunits has created 2 sites at which aminoacyl tRNAs can bind

Elongation involves the next amino acid binding to the A-site and then being moved long to the P-site where is can be joined to the growing peptide chain (so far the “chain” is only one aa – the methionine- but it’s going to get bigger).

Question 15

What are the 3 elongation factors?

Answer 15

EF1A (EF-Tu)
Loads aa-tRNA into the A-site. Commonest protein in cell.
EF-1B (EF-Ts)
Recycles EF-Tu
EF-2 (EF-G)
Checks that translocation is successful

Question 16

What happens during elongation in translation?

Answer 16

1) 2 parts of the ribosome are now together= P-site is already occupied by initiator tRNA
2) Next aminoacyl-tRNA will bind to A site
3) EF-1A protein in bacterial cell and complexes with GTP and aminoacyl-tRNAs= pool of these in each cell and ONLY the correct amino acid can load their amino acid into the empty A-site
4) Matching aminoacyl-tRNA is loaded, EF1-A will hydrolyse GTP= Release energy to allow formation of a peptide bond by the ribozyme peptidyl transferase
5) EF-1B recycles EF-1A by removing GDP and allow free GTP to bind, which can now pick up new aa-tRNA
6) Ribosome must now move 5’ to 3’ 3 bases to move the next codon into A-site, aided by EF-2 which hydrolyses GTP upon successful translocation

Question 17

What happens during translocation (elongation)?

Answer 17

First 2 amino acids have been joined= Attachment between first amino acid and its aminoacyl tRNA are broken

Translocation= Ribosome moves 3 nucleotides along mRNA= ejecting deacylated initiator tRNA from P site= Now occupied by 2nd tRNA attached to first 2 amino acids of the polypeptide

New codon can now enter A site

Translocation= hydrolysis of a molecule of GTP and is mediated by EF-2

Ejected aminoacyl tRNA leaves via 3rd site= Exit site

Ribosome can slide along the mRNA so it repositions itself each time a new amino acid needs to be added

Question 18

Where does the synthesis of the peptide bond occur?

Answer 18

P-site

Peptide transferase enzyme

The peptide bond is synthesised by the transfer of the growing polypeptide chain from the P-site tRNA to the free amino group on the amino acid on the tRNA in the A-site.

Question 19

What are releasing factors?

Answer 19

Proteins that mimic aminoacyl-tRNA and can load into the A-site

RF-1: Recognises UAG and UAA

RF-2: Recognises UGA and UAA

RF-3: Stimulates dissociation of RF-1 and RF-2

Question 20

What happens during phase 3? (Termination)

Answer 20

1) Termination codon (UAA, UGA or UAG) moves into A-site= signal for ribosome to release the now complete protein
2) Releasing factors loads onto A site (Either Rf-1 + RF-3 OR RF-2 + RF-3) and bond between the last amino acid of the protein and the adenine of the tRNA in the P-site is hydrolysed
3) Releases the protein as it tries to make another peptide bond but there is no amino acid in the A-site= peptide is transferred not to the amino group of an amino acid but to water
4) Ribosome is separated into its 2 subunits by the ribosome recycling factor (RRF)

Question 21

What happens when you alter each base in the Genetic code?

Answer 21

Altering 3rd base= Usually no effect

Altering 1st base= Will change one amino acid to a biochemically similar one

All in the first column= Hydrophobic amino acids

All in second column=
Polar amino acids

Many in 3rd column are basic or acidic

Question 22

What does the ‘wobble’ term describe?

Answer 22

The fact that one tRNA can recognise more than one codon for the same amino acid if the codon differs only in its 3rd position (3rd base)

Example: Can recognise GCC or GCU