In a theoretical operon, the genes A, B, C, and D represent a repressor gene, a promoter sequence, an operator, and the structural gene (enzyme) but not necessarily in that order. The enzyme encoded by this operon is involved in the synthesis of a theoretical molecule (TM). You are not sure whether this operon is induced by the TM precursor (similar to the lac operon) or repressed by the product (like the trp operon). Your assay is whether or not the enzyme is transcribed, and whether it is active. Using the chart below, determine whether the operon is inducible or repressible, and then assign A, B, C, and D to the four parts of the operon. In the chart, AE = active enzyme; IE = inactive enzyme; NE = no enzyme present). Note that the F' plasmids contain more than one WT gene, and there are two different versions. Medium supplemented with:
Genotype: F' genotype nothing TM precursor TM
A+B+C+D+ (none) AE AE NE
A-B+C+D+ (none) NE NE NE
A+B-C+D+ (none) AE AE AE
A+B+C-D+ (none) IE IE NE
A+B+C+D- (none) AE AE AE
A-B+C+D+ F' A+B+D+ NE NE NE
A+B-C+D+ F' A+B+D+ AE AE AE
A+B+C-D+ F' A+B+C+D+ IE +AE IE+AE NE
A+B+C+D- F' A+B+D+ AE AE NE
Using these data, answer the following questions:
1. Is the TM operon inducible or repressible? WHY?
2. Which molecule acts as the inducer or corepressor? WHY?
3. Indicate which sequence (A, B,C, or D) encodes each of the following and explain why you think so:
Promoter:
Structural gene for enzyme:
Repressor gene:
Operator:
4. Why did we leave C+ out of the F' plasmid in most cases? Why would it have complicated the interpretation of the results?
5. What is the nature of the repressor mutation? Can we tell from these data whether the mutation (1) prevents binding to operator; (2) prevents binding to corepressor/inducer or (3) prevents release from operator and HOW?