Gene Expression

Question 1

What is a gene?

Answer 1

Stretch of DNA with a chromosomal locus, representing:

• a unit of inheritance
• a unit of transcription.

Question 2

What are the 5 different types of RNA?

Answer 2

1. mRNA - 2%
   
   • 100,000s kinds
   • few copies of each
2. rRNA
   
   • 4 kinds
   • many copies of each
3. tRNA
   
   • 100 kinds
   • very many copies of each
4. miRNA
5. Non-coding RNA

Question 3

Where do transcription and translation occur?

Answer 3

• Transcription = nucleus

- Translation = cytoplasm

Question 4

Describe the requirements of transcription and translation in terms of: enzyme, activated substrates, template and process.

Answer 4

Enzyme: RNA polymerase vs ribosome

Substrates: NTPs vs amino acids

Template: DNA vs mRNA

Process: initiation, elongation and termination.

Question 5

Describe the initiation stage of transcription.

Answer 5

1. TFs bind to gene promoter region (TATA box).
2. Recruit RNA polymerase II. RNAP binding/activity requireds presence of:
   
   • activator
   • mediator
   • chromatin-remodelling complex
   • histone acetylase
3. TF helicase unwinds DNA - exposes a DNA coding strand and DNA template strand.

Question 6

Describe the elongation stage of transcription. In which direction does synthesis occur?

Answer 6

4. RNAP moves along the template strand (reads 3’ to 5’), covalently linking nucleotides to form pre-mRNA strand (synthesises 5’ to 3’).
5. Elongation is coupled to RNA processing.

Question 7

Describe the termination stage of transcription.

Answer 7

6. RNAP reaches 3’ termination sequence and falls of template strand.

Question 8

How is pre-mRNA turned into mature mRNA?

Answer 8

Post-transcriptional processing:

• 5’ RNA capping
• 3’ polyadenylation
• intron splicing

Question 9

What is 5’ capping and why does it occur?

Answer 9

• 7-methyl guanosine cap is added to 5’ end via 5’-5’ phosphate linkage.
• protects against degradation and role in translation initiation.

Question 10

What is 3’ polyadenylation and why does it occur?

Answer 10

• Specific endonuclease recognises AAUAA cleavage site and cleaves mRNA a few bases further.
• Poly-A polymerase adds up to 200 A nucleotides.
• Protects against degradation.

Question 11

Which enzyme catalyses the translation reaction?

Answer 11

Ribosomes

• 2 subunits
• made up of 4 rRNAs + 82 proteins

Question 12

What is a codon?

Answer 12

• Nucleotide triplet in mRNA that encodes the information for a specific amino acid in a protein.
• Recognised by a complementary tRNA anti-codon.

Question 13

Why is the triplet code degenerate?

Answer 13

Each codon (64) codes for 1 or more of the 20 aa.

Question 14

What are the start and stop translation codons?

Answer 14

Initiation = AUG (methionine)
Termination = UAA, UAG, UGA

Question 15

What is the wobble hypothesis?

Answer 15

• 1 tRNA anti-codon recognises >1 codon: although pairing at first 2 bases must be complementary, pairing at 3rd codon base doesn’t have to match (‘wobbles’).
• Accommodated for by presence of inosine base: can recognise U, C or A.

Question 16

Describe the structure of a tRNA molecule.

Answer 16

• Forms clover shape of stem loops - hydrogen bonds are formed between anti-parallel, complementary sequences.
• Contains 2 attachment sites:
  
  1. Anti-codon that pairs with complementary mRNA codon
  2. Amino acid attachment site

Question 17

How is an amino acid attached to tRNA?

Answer 17

Via aminoacyl-tRNA synthetase (requires ATP and releases 2 P).

Question 18

Describe the initiation stage of translation.

Answer 18

1. Initiator complex assembles on 5’ cap (cap binding proteins, initiation factors, 40S ribosome subunit, Met-tRNA and GTP).
2. Methionine-tRNA scans mRNA and finds AUG start codon.
3. GDP, initiation factors and cap binding proteins are removed from complex whilst 60S ribosome subunit is recruited.

Question 19

Describe the elongation stage of translation.

Answer 19

4. Met-tRNA binds to AUG codon (GTP to GDP + Pi) in ribosome P site.
5. Next complementary amino-acyl tRNA moves into ribosome A site and Met is transferred to second tRNA via peptide bond formation (peptidyl transferase) between the amino acids.
6. P site uncharged tRNA exits the ribosome and the ribosome translocates (GTP to GDP + Pi) so that 2nd peptidyl-tRNA is in P site.
7. Newly charged tRNA enters A site etc.

Question 20

Describe the termination stage of translation.

Answer 20

8. Ribosome encounters a stop codon in the mRNA.
9. Release factor binds to stop codon and catalyses hydrolysis of polypeptide from tRNA.
10. Polypeptide, mRNA and tRNA are released from the ribosome.