Protein Synthesis

Question 1

How did Hoagland, Stephenson, Scott, Hecht and Zanecnik show that free soluble rnase brought amino acids to the ribosomes for protein synthesis (helping support discovery of tRNA)

Answer 1

Hoagland, Stephenson, Scott, Hecht and Zamecnik 1958.
1. Separated ribosomes from cytoplasm soluble RNA molecules.
2. Used differential centrifugation (ribosomes can’t synthesis unless mixed with cytoplasmic rnase)
3. Labelled lucine with C14
4. Radioactivity went to purified small RNA
5. Put ribosomes and small RNA back together
Radioactivity was lost from small RNA but found in newly synthesised protein

Question 2

Structure of tRNA

Answer 2

• single RNA strand ~80nt long
• flattened representation (clover leaf structure)- reveals base pairs
• 3’ and 5’ ends come together
• hydrogen bonds fold it into a 3D L shape

Question 3

How do isoacceptors help solve degeneracy of the genetic code

Answer 3

Recognition that there’s 20 aminoacyl-tRNA transferase enzymes

Question 4

What are the binding sites in the ribosome

Answer 4

A bind site
P binding site
E binding site

Question 5

What are the 3 stages of translation

Answer 5

1. Initiation
2. Elongation
3. Termination

Question 6

What do the regulatory factors of translation require

Answer 6

GTP and ATP

Question 7

How are ribosomes assembled and how is initiation of translation initiated

Answer 7

1. Small ribosomal subunit binds to mRNA
2. Subunit slides until it finds AUG
3. Large ribosomal subunit binds = initiation complex
4. Met-tRNA at the P site

Question 8

When is the input of elongation factors and GTP hydrolysis needed

Answer 8

When the initiation complex is formed - GTP hydrolysis

Amino acid addition - elongation factors (proteins)

Question 9

In which direction does growth of the polypeptide go

Answer 9

In the direction of the carboxyl end.
New amino acid is linked to 3’ hydroxyl end of tRNA by carboxyl group.
Leaves 1 amino acid group to establish a peptide bond.

Question 10

In what stages of translation is energy expended

Answer 10

Codon recognition and translocation

Question 11

What occurs during codon formation

Answer 11

1. Codon recognised by Met-tRNA
2. Met-tRNA anchored to ribosome using GTP
3. Met-tRNA is stabilised

Question 12

How are peptide bonds formed in the synthesised polypeptide chain

Answer 12

Between the carboxyl end and the amino group.

No extra energy cost.

Question 13

What occurs during translocation

Answer 13

1. 2 tRNA and 2 mRNA move towards their E site
2. Empty tRNA reaches E site and exits
3. A site tRNA moves to P site holding the polypeptide chain
4. Free A site to display codon

Question 14

How is translation terminated

Answer 14

1. Ribosome reaches a stop codon on mRNA
2. A site accepts the ‘release factor’ protein
3. Promotes a hydrolysis reaction, freeing the polypeptide from tRNA in P site
4. Ribosome subunits and components disassemble

Question 15

What is transcriptional regulation

Answer 15

Signal reaching chromatin, chromatin modification: DNA unpacking, gene available for transcription

Question 16

What is post-transcriptional regulation

Answer 16

Transcription, RNA processing, transport to cytoplasm, translation and protein processing

Question 17

What is post-translational regulation

Answer 17

Degradation of protein and transport to cellular destination (to keep out the way until it’s needed)

Question 18

How can translation be blocked

Answer 18

Regulatory proteins - bind to 5’ UTR of mRNA - modulating amount of protein produced
Promote introduction of mRNA with ribosomes - make polyribosomes or polysomes (synthesis of many proteins as lots of ribosomes attach to mRNA)

Question 19

What does the lifespan of mRNA tell us

Answer 19

How much protein is produced

- achieved by promoting mRNA degradation

Question 20

How do you calculate the lifespan of mRNA

Answer 20

Nucleotide sequences that affect mRNA longevity are found in the 3’ untranslated region, following stop codon

Question 21

How is mRNA degraded

Answer 21

1. Enzymes attack the poly-A-tail

2. Enzymes eventually attack the rest of the mRNA

Question 22

Roughly how long is the lifespan on mRNA

Answer 22

Hours - weeks. Depends on the ability to resist degradation

Question 23

The role of 5’ UTR in mRNA

Answer 23

Influence mRNA interaction with the ribosome

Question 24

The role of 3’ UTR in mRNA

Answer 24

Influence mRNA stability/life span (stability influence protein synthesis)

Question 25

The role of the 5’ cap in mRNA

Answer 25

Important for initiation of translation

Question 26

Give an example of when you may need to stop or modify protein activity

Answer 26

Embryonic development
- embryo with divide for the last time the differentiate - cell needs to stop the function of the proteins, can’t switch off and wait for mRNA and proteins to disappear, so much inactivate or destroy them.

Question 27

How do you quickly destroy a protein

Answer 27

Send it to a proteasome - breaks it down

Question 28

Strategies to control protein activity post-translationally

Answer 28

Regulating protein localisation

Question 29

How can ribosomes inject a nascent polypeptide into the lumen of the ER

Answer 29

If bound ribosomes are located with p sites oriented towards the entrance of the ER channel

Question 30

How do you export a protein from a cell

Answer 30

1. Polypeptide synthesis begins
2. SRP binds to signal peptide
3. SRP binds to receptor protein (on the ER)
4. SRP detaches and the polypeptide synthesis resumes
5. Signal cleaving enzyme cuts off signal peptide
6. Completed polypeptide folds into final conformation

Question 31

What is the effect of adding lateral chains of amino acids to different molecules (methyl, acetyl, lipids or small proteins)

Answer 31

• they’re specific to each protein
• can modify it positively
• can modify it negatively
• affect activity, stability to localisation of the protein

Question 32

What does translation produce

Answer 32

A polypeptide NOT a protein