The soluble and membrane-anchored isoforms of Mgm1 are only active when they work together (in trans).

SummaryMlc and NagC are two homologous transcription factors which bind to similar DNA targets but for which the inducing signals and mechanisms of activation are very different. Displacing Mlc from its DNA binding sites necessitates its sequestration to the inner membrane via an interaction with PtsG (EIICB Glc ), while NagC is displaced from its DNA targets by interacting with GlcNAc6P. We have isolated mutations in both proteins which prevent the inactivation of the repressors by growth on glucose or GlcNAc. These mutations are located in different and specific regions of each protein. For Mlc changes at the C-terminal make it a constitutive repressor and also prevent it from binding to EIIB Glc . Mutations in NagC, at positions which form a structural motif resembling a glucose binding site in Mlc, produce permanently repressing forms of NagC, suggesting that this motif forms a GlcNAc6P binding site in NagC. The pattern of repression by chimeric proteins of NagC and Mlc confirms the importance of the C-terminal region of Mlc for both repression and inducer binding and demonstrate that the helix-turn-helix DNA-binding motif is not sufficient to determine the specificity of interaction of the repressor with DNA.

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Summary Protein–DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N‐acetylglucosamine. Both their helix–turn–helix motifs and their consensus binding sites on DNA are very similar. One distinguishing feature is that most NagC sites have a C/G bp at positions −11 and +11 from the centre of symmetry of the operator, while all Mlc sites have A/T. By constructing Mlc and NagC chimeras, we show that the helix–turn–helix motif per se is not responsible for specific recognition of Mlc or NagC sites, but that a linker, joining the DNA‐binding domain to the rest of the protein, is the major determinant. We show that a change of just two amino acids in the NagC linker is sufficient to allow NagC to recognize an A/T bp at positions +/−11 and repress Mlc targets. Modelling of the NagC linker suggests that it forms an extended structure containing two arginines and we suggest that these arginines interact differently with the minor groove at positions +/−11 depending upon the presence of a C/G or A/T bp.