Question

Negative regulation in eukaryotes can occur by direct or indirect repression. You have isolated a protein involved in negative regulation and are using a system with GFP attached as a reporter construct. Show how you might use this system to see whether competition or quenching negative regulation is occurring. First, please define these types of negative regulation in your answer.

Answer 1

Answer 1)

1. Negative inhibition of gene transcription can be brought out by binding of a repressor protein molecule to the operator sequence which in turn prevents RNA Polymerase from initiating the process.
2. In competitive negative inhibition, there is competition between the inhibitor or the repressor molecule along with the substrate molecule to occupy the active site and depends upon the conformational changes occuring within the enzyme molecule to inhibit the reaction.
3. In quenching negative regulation, it is defined in terms of quorum sensing pathway. In this pathway, the signalling molecules are involved in generating a gene transcriptional response on the basis of cell density. These signalling molecules are disturbed by presenting a similar molecule that mimics the molecule and occupies the receptor position that seems to be the target for the original signalling molecule.

Answer 2)

1. The gene of interest which needs to be studied can be kept under the control of a strong promoter such as Cauliflower Mosaic Virus Promoter downstream of which the sequence for Green Fluorescent Protein is inserted. This is followed by the target DNA sequence.
2. This construct can then be cultured in-vitro and checked for expression process.
3. So, the first step is tagging of GTP followed by insertion into cells.
4. The endogenous protein may be allowed to express and other batch may be treated with repressor protein with particular mechanism. For study of competitive repressor, concentrations needs to be monitored. In term of quenching inhibition, specific protein should be added in terms of signalling and receptor binding to study the mechanism and to allow binding of homolog to inhibit the reaction.
5. The luminescence can be measured by means of luminometer.
6. This corresponds to the enzyme activity and rate of transcription for data analysis purpose.