Question

1. Antibody is prepared to the thr repressor protein isolated from a wild-type strain.  Competetive antibody binding assays show that several times more repressor is present in an extract prepared from the derepressed mutant described above than in an extract prepared from the wild-type strain.  What is your interpretation of these data?
2. The repressor protein requires a pH of 6.1 for optimal binding to its operator in vitro.  If the pH goes above pH 8.0, the repressor is unable to bind.  What amino acid side chains are implicated?  Why is binding reduced in alkaline media?
3. What effect would an increase in pH have on cell transcription of the thr operon? Why?
4. What percentage of the repressor will bind at pH 6.3?  More than 50% or less?  Now calculate.

Answer 1

a.
Derepressed mutants will allow the increased expression of the repressor. Hence for activation of the operon, more concentration of Thr (threonine) is required. Because, Thr will bind to the repressor and will not allow it to bind to the operator and hence operon will be ON. However, in the derepressed mutant presence of the more repressor will require more concentration of Thr to keep it ON state as compared to the wild type.

b.
Cysteine side chain has a isoelectric pH of the 8.37, at pH above 8.3 cysteine will have negative change. Operator is the DNA and hence it is negatively charges. Therefore, negative charge of the repressor at pH 8 will not allow the binding of the repressor to the negatively charged operator DNA.

c.
Transcription will be increased at increasing pH above 8. As explained in b, presence of negative charge of the repressor will not allows the binding of repressor to the operator DNA. Therefore, their will be no hurdle for the movement of the RNA polymerase on the DNA.

d.

More than 50% will bind to the operator DNA, because at pH 6.3 charge on the repressor will be same as that at pH 6.1. Isoelectric pH of the cysteine side chain is 8.3. Therefore, for all the pH below 8.3 cysteine will have the same charge. The charge on the cysteine side chain at pH below 8.3 would be neutral because -SH group will not be protonated further to add positive charge. Hence, the total charge of the repressor at pH below 8.3 will be decided by protonation and deprotonation state by other charged amino acids present. However, at pH above 8.3 cysteine add negative charge to the total charge of protein.