You started work in a transcription lab working a with a novel bacteria phage. Y

Question

You started work in a transcription lab working a with a novel bacteria phage. You isolated an enzyme that has RNA polymerase activity, and you discovered that your new viral RNA polymerase has a unique property. Unlike E. coli RNA polymerase, which prefers starting transcription with ATP, your new viral polymerase prefers starting transcription with GMP.

A) Explain in what direction (3’-to-5’ or 5-to-3’) this enzyme synthesizes RNA and how you know this.

B) What other simple experiment can you do to show that your conclusion is correct, provided you have full access to all rNTPs, 3’-dNTPs, 2’-dNTPs?

Explanation / Answer

Please find the answers below:

Answer A: Transcription or synthesis of RNA takes place universally in the 5' to 3' direction irrespective of the nature of source of energy or other biomolecules. The direction of transcription is always 5' to 3' since the enzyme RNA polymerase recognizes only the nucleotides located in this direction on the template DNA irrespective of the other ingredients for transcription to take place. The presence of incomplete mRNA strands in growing 3'direction on an autoradiogram using Sanger's method can help in demonstrating this fact.

Answer B: In order to conclude it, radiolabelled or fluorphore labelled dNTPs can be added to the reaction mixture. The reaction mixture is allowed to undergo transcription for a small fraction of time and after a few intervals, the sample is taken out and resolved onto gel. The gel is exposed to fluorescence excitation or scintillator. The sequence is monitored, linearly arranged and matched to that of the parent DNA molecule. This would reveal that transcription indeed takes plance in 5' to 3' rection.

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