Experiments to identify the position of the promoter on a plasmid and the direction of transcription Flashcards Preview

BS2091 Biochemistry I: from genes to proteins > Experiments to identify the position of the promoter on a plasmid and the direction of transcription > Flashcards

Flashcards in Experiments to identify the position of the promoter on a plasmid and the direction of transcription Deck (13):
1

What type of molecule is a plasmid?

A small, circular molecule of DNA that can be replicated separately from the chromosomal DNA in a cell

2

Where are plasmids commonly used and what for?

Commonly used in molecular biology for cloning and heterologous protein expression

3

What can the expression of given polymerases be put under the control of?

The lac operon

4

What does the lac operon consist of?

The lac promoter
The lac operator
The lac I (repressor protein)

5

What does the addition of IPTG do when added to the medium of cells containing plasmid?

It binds to the lac repressor protein and releases the repressor from the lac operator, allowing the transcription of genes in the lac operon by the cell's RNA polymerase

6

What would a produced polymerase need to have?

A ribosome binding site (RBS) for protein translation
A start methionine for initiation
A 6x histidine tag for protein purification

7

Briefly define affinity chromatography

A powerful method to purify a protein of interest, from other proteins and components in a crude cell lysate, based on its specific binding properties to an immobilised ligand

8

Affinity chromatography can be very useful when..

Recombinant proteins are expressed with affinity tags

9

How recombinant His-tagged proteins be purified?

Affinity chromatography using Ni-NTA agarose
Ni-NTA agarose has immobilised nickel ions that bind the histidine residues in the tag
His-tagged proteins bind with high specificity and affinity and contaminating proteins pass through the resin
After washing, His-tagged proteins are eluted with a buffer containing imidazole

10

What is T7 RNA polymerase (T7 RNAP)?

A DNA-dependent RNA polymerase from T7 bacteriophage

11

What does T7 RNAP do?

Catalysed the formation of RNA in the 5' to 3' direction

12

Is the T7 polymerase promoter sequence specific on non-specific for this enzyme?

Very specific
(The promoter sequences are conserved from positions -17 to +3 relative to the start site of transcription)

13

How is the transcription of the T7 RNAP terminated?

By either an RNA hairpin, known as the transcription terminator or, in vitro, by prior cleavage of the DNA with a restriction enzyme such that the template comes to an end, known as a 'run-off' transcription assay