RNA Pol II

Question 1

A gene can have more than one promoter. Give and example of such a gene. What are the terms for each of these promoters?

Answer 1

LCK (lymphocyte protein tyrosine kinase)

Proximal and Distal promoters

Question 2

How far away is the distal promoter from the proximal promoter for the LCK gene?

Answer 2

22kb

Question 3

How can you find which specific region of the promoter is responsible for Pol II binding?

Answer 3

Reporter Assays and Saturation Mutagenesis

Question 4

Give the key features of reporter assays

Answer 4

1. Reporter must be measurable (e.g. An enzyme such as luciferase or beta-galactosidase)
2. They must be contained on a plasmid backbone

Question 5

How does a luciferase reporter assay work?

Answer 5

Fuse gene of interest with luciferase gene. Transiently transfect cells, e.g. HeLa cells. Express. Add luciferin, Mg and ATP. Measure using a ‘luminometer’

Question 6

What two techniques can be combined to determine sequences in the promoter required for transcriptional activity?

Answer 6

Saturation mutagenesis and reporter assays. Then can identify binding proteins with gel shift/ mobility assay

Question 7

Approximately where are the GC box, CAAT box and TATA box located in a sequence of DNA, and which protein binds to each?

Answer 7

GC box: -100 bp: SP1 (specificity protein 1)
CAAT box: -80 bp: NF1 (nuclear factor 1)
TATA box: -30 bp: TBP (TATA binding protein)

Question 8

What are the advantages of using a cDNA library over RNA?

Answer 8

1. DNA is more stable than RNA
2. Can sub-clone into plasmids and sequence
3. cDNA clone for every RNA transcript in cell