Direct and indirect readout in mutant Met repressor-operator complexes

Background: The methionine repressor, MetJ, represses the transcription of genes involved in methionine biosynthesis by binding to arrays of two to fi ve adjacent copies of an eight base-pair 'metbox' sequence. Naturally occur ring operators differ from the consensus sequence to a greater extent as th e number of metboxes increases. MetJ, while accommodating this sequence var iation in natural operators, is very sensitive to particular base changes, even where bases are not directly contacted in the crystal structure of a c omplex formed between the repressor and consensus operator. Results: Here we report the high-resolution structure of a MetJ mutant, Q44 K, bound to the consensus operator sequence (Q44Kwt19) and two related sequ ences containing mutations at sites believed to be important for indirect r eadout at non-contacted bases. The overall structure of the Q44Kwt19 comple x is very similar to the wild-type complex, but there are small Variations in sugar-phosphate backbone conformation and direct contacts to the DNA bas es. The mutant complexes show a mixture of direct and indirect readout of s equence variations, with differences in direct contacts and DNA conformatio n. Conclusions: Comparison of the wild-type and mutant repressor-operator comp lexes shows that the repressor makes sufficiently strong interactions with the sugar-phosphate backbone to accommodate some variation in operator sequ ence with minor changes in direct bases contacts. The reduction in represso r affinity for the two mutant repressor complexes can be partially attribut ed to a loss in direct contacts to the DNA. In one case, however, the repla cement of a flexible TA base-step leads to an unfavourable DNA conformation that reduces the stability of the repressor-operator complex.