Question

You have isolated a piece of DNA that you believe contains an interesting gene. Using a mix of RNA polymerase and ribonucleotides, you perform in vitro transcription. However, even though all of your controls work, no mRNA is created from your DNA fragment. Working backward, you note that your DNA preparation removed approximately 250 base pairs from the 5' end of the gene. The most likely explanation is that during DNA preparation?

1) the termination signal was removed.
2) the RNA polymerase became inactivated.
3) the 5' methyl guanosine cap was removed.
4) the promoter was removed.
5) None of these explains the negative results.

Answer 1

The absence of mRNA following in vitro transcription is most likely due to the removal of the promoter region from the DNA template during the preparation process. This region is crucial for RNA polymerase to attach and begin transcription.

Step-by-step explanation:

If you have isolated a piece of DNA for in vitro transcription and find that no mRNA is created even though all controls work, and you note that about 250 base pairs from the 5' end of the gene were removed during DNA preparation, the most likely explanation is that the promoter was removed. The promoter region is essential for RNA polymerase to bind and initiate transcription. Without it, the enzyme cannot recognize where to start and thus no transcription occurs.

In prokaryotic systems, transcription initiation does not require a 5' methyl guanosine cap, which is a feature of eukaryotic mRNA. The termination signal, which includes rho-dependent termination and rho-independent termination, is at the 3' end of the gene and would not prevent initiation. It is also unlikely that RNA polymerase became inactivated if the controls are functioning correctly.

Therefore, the correct answer to why no mRNA was created from the DNA fragment is likely because the promoter was removed during DNA preparation, corresponding to option 4 of the provided choices.