Question

You include an innhibitor of the protein kinase activity of TFIIH in an in virto transcription. What step in transcription would you expect to see blocked? Decribe an experiment you would run to test your hypothesis?

Please explain the experiment part to the best of your ability.

Answer 1

Carboxylic terminal domain (CTD) of a RNA polymerase ll undergoes dynamic phosphorylation during transcription cycle.

Phosphorylation of CTD is required for futher steps like promoter clearance and elongation of transcript.

For all these above steps protein kinase of TFIIH is required.

TFIIH is a multiprotein complex which is required for both transcription and DNA repair.

It mainly involves in three functions :

1) phosphorylation of carboxylic end (-COOH) of terminal domain (CTD) of RPB1 subunit of RNA polymerase II.

2) promoter melting

3) promoter clearance

This shows that the inhibition of protein kinase activity of TFIIH leads to blockage of elongation.

The experiment to the test the above hypothesis is G-less cassette assay.

It mainly used to determine the promoter strength.

The above test is usually used to synthesis the transcript of predicatable size using polyacrylamide gel.

In this experiment promoter is fused to double stranded DNA lacking G's in non template strand.

Transcription process undergoes in absence of GTP. Transcription aborts at end of cassette where G is required and produces aborted transcripted of predicatable size.

The formed aborted transcripts electrophoresed and gel is autoradiographed. Based on promoter strength number of aborted transcripts are produced.

But in the above case when inhibitor is added transcription elongation is blocked and there will be sudden decrease in the labelled aborted transcripts.