DD - Transcription I

Question 1

Why is an intermediate mRNA required in the DNA to protein process? (3)

Answer 1

1) Better regulation of the protein production process

2) Fidelity of DNA replication is very important

• Having an intermediate step prevents interference and potential additional mistakes.

3) Proteins can still be made when DNA is being replicated

Question 2

What is RNA? (4)

Answer 2

1. RNA is a linear polymer like DNA
2. Residues linked by phosphodiester bonds
3. Contains ribose not deoxyribose
4. RNA is single-stranded

Question 3

What are the main types of RNA and their functions?

Answer 3

rRNA: Ribosomal RNA

• Form the basic structure of the ribosome and catalyse protein synthesis

tRNA: Transfer RNAs

• Central to protein synthesis as adaptors between mRNA and amino acids

Non-coding RNA

• e.g. microRNAs, long non-coding RNAs

mRNA: Messenger RNA

• Codes for proteins (3-5%)

Question 4

What are 6 features of RNA Polymerase?

Answer 4

• Crab claw
• Separates the 2 strands of DNA
• Uses one of the DNA strands as a template for RNA synthesis
• Does not require a primer
• It is very accurate 1/10,000 bases
• Moves along the gene in a 5’ to 3’ direction

Question 5

What are the key steps in transcription?

Answer 5

1. Initiation - Template recognition

a) Polymerase locates promoter

b) Polymerase unwinds the DNA

c) Transcription begins

2. Elongation

a) Polymerase places RNA in exit hole

b) Once RNA is 10 or more base pairs long

c) Polymerase conformation change - tightens grip

3. Termination

a) Termination sequence

b) Polymerase separates

c) RNA released

Question 6

What is the consequence of protein over-expression in Escherichia coli?

Answer 6

Protein over-expression in Escherichia coli can trigger an adaptation process similar to antimicrobial resistance

Question 7

Check slides for the E.coli pET over-expression system

Answer 7

Plasmid Preparation: Design a plasmid containing the target gene under the control of the T7 promoter.

Host Strain Selection: Choose an E. coli host strain for transformation.

T7 RNA Polymerase Introduction: Transform the host strain with a separate plasmid carrying the T7 RNA polymerase gene under the control of a lac promoter.

Transformation: Introduce the target gene plasmid into the host strain.

Induction: Add the inducer, typically IPTG, to the E. coli culture to activate the T7 promoter and initiate gene expression.

Transcription: T7 RNA polymerase transcribes the target gene into mRNA.

Translation: Ribosomes in the E. coli cells translate the mRNA into the target protein.

Protein Production: Overproduction of the desired protein.

Harvest and Purification: Collect the cells and purify the target protein from the lysate for various applications