You would like to transcribe a eukaryotic gene in vitro (ie. in a test tube in t

Question

You would like to transcribe a eukaryotic gene in vitro (ie. in a test tube in the lab). Your goal is to achieve a high level of transcription. To your test tube, you add the DNA template for this gene, which contains the following: a TATA box sequence located –25 to –30 bp upstream of the transcription start site, the sequence for the gene, and terminator sequences. You also add RNA polymerase and the transcription factors that make up the basal transcriptional machinery. However, after doing your experiment, you do not get very high levels of transcription. What do you think might be the cause of this and what corrective measures would you take to increase the level of transcription?

Explanation / Answer

In the above test tube, there is presence of general transcription factors, TATA sequence, transcription site, terminator sequence, RNA polymerase. However, the levels of transcription obtained are not very high. This is because, there is a missing activator sequence in the test tube. Also, along with the activators, there requires to be a co activator sequence. The CO activator is a sequence that would be working along with the transcription factor so as to increase the gene transcription rate.

In this case, we require to add a CO activator sequence along with an activator sequence. The conactivator would be binding to the activator so as to bind to the promoter site. Also, there requires to be a site named as enhancer. The activator and CO activator complex would increase the transcription rate as it would be recruiting the General transcription machinery.

Since it is a eukaryotic gene, all the three RNA polymerase shave it be added and not one.

High levels of transcription would be achieved only when the enhancer sequences would be present for transcription. These sequences would increase transcription and achieve high levels of transcription when coupled with CO activators and activators. The interaction of enhancers and the DNA sequence will also be mediated by the sequences called mediators. These increase the frequency as well as efficiency of the basal complex of transcription that would be formed at the promoter. Enhancer sequences are required so that they could bind to different transcription factors and it varies the transcription factor combinations regulating transcription.

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