After performing a Lys operon mRNA Northern blot and Western blot of the two oth

Question

After performing a Lys operon mRNA Northern blot and Western blot of the two other proteins expressed from the same mRNA, LysD and LysE, your results are as follows

You proceed to determine the sequence of the entire mRNA and its structure is as below

I = Lys repressor gene; D, E, F = Lys operon structural genes; P = Lys promoter; O = Lys operator
An outsourced advisor assures you that the type of the regulation exhibited by this operon is transcriptional attenuation. You disagree with him (as polite as possible) based on the observations from the blots above. What observation is there to justify your point?

Explanation / Answer

In transcriptional attenuation, there is premature termination of transcription. Hence, the corresponding mRNA is not produced for synthesis of the remaining proteins.

Lys D and LysE are structural genes that are present in the same lac operon. Hence, the same mRNA should code for both genes. The operon is stimulated when amino acids are reduced or absent in the medium.

The mRNA transcription is not increased when amino acids are added to the medium. Hence, amino acids do not have an effect on transcription. The rate of mRNA synthesis remains the same, as depicted by the Northern Blot. The Northern blot indicates presence of mRNA. Hence, mRNA synthesis is not abrogated by removal of amino acids. If transcription was prevented, then no transcript (mRNA) would be seen upon removal of the amino acids, as the probes are complementary to the operon.

The lack of amino acids is also able to stimulate production of the proteins LysD and Lys E (western blot). The genes for these proteins are present at the end of the operon. The increase in synthesis of the proteins may not be attributed to increased mRNA synthesis. Other stimulatory mechanism may be involved, that are post transcription (increased mRNA stability) or post translational (phosphorylation etc). If transcriptional attenuation were the reason for the regulation, no mRNA, LysD and lys E proteins would have been detected in the blots, as the mRNA would lack the genes coding for these two proteins. The probe in Northern blot would not anneal with the mRNA for detection when amino acids were absent in the medium.

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