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Homologue function can be differentiated by changing residues that affect binding sites or long-range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA-binding and regulatory domains linked by approximately 18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be the cause of altered function or an effect of the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA-binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue-specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression from lacO1 and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR-like interactions via substitutions in the LLhP linker tends to diminish repression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for the lacO1 DNA site is sensitive to long-range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co-evolved with changes in the DNA-binding site. In addition, repression measured in the absence and presence of effector ligand shows that allosteric response increases for several LLhP variants with substitutions at positions 48 and 55. Thus, while side chain variation at these sites does not generally dictate the presence or absence of allostery, the nature of the amino acid can modulate the response to effector.

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The sequencing of the EcoRI-HindIII fragment complementing mutations in the structural genes of the L-rhamnose regulon of Escherichia coli has permitted identification of the open reading frames corresponding to rhaB, rhaA, and rhaD. The deduced amino acid sequences gave a 425-amino-acid polypeptide corresponding to rhamnulose kinase for rhaB, a 400-amino-acid polypeptide corresponding to rhamnose isomerase for rhaA, and a 274-amino-acid polypeptide corresponding to rhamnulose-1-phosphate aldolase for rhaD. Transcriptional fusions of the three putative promoter regions to lacZ showed that only the rhaB leader region acted as a promoter, as indicated by the high 13-galactosidase activity induced by rhamnose, while no significant activity from the rhaA and rhaD constructions was detected. The rhaB transcription start site was mapped to -24 relative to the start of translation. Mutations in the catabolic genes were used to show that L-rhamnose may directly induce rhaBAD transcription.L-Rhamnose, a methylpentose, is metabolized in Escherichia coli by a set of enzymes encoded by genes constituting the rhamnose regulon, which maps at 88.4 min in the chromosome (2). Four structural genes have been described: rhaA, encoding rhamnose isomerase; rhaB, encoding rhamnulose kinase; rhaD, encoding rhamnulose-1-phosphate aldolase (32); and rhaT, encoding the rhamnose transport system (17). The rhaT gene has been mapped in the rha locus, separated from rhaA, rhaB, and rhaD by the regulatory operon rhaC, which has been found to be formed by two partially overlapping genes, rhaR and rhaS (40). The gene order of the region, counterclockwise, is glpK... sodArhaT-rhaR-rhaS-rhaB-rhaA-rhaD.In E. coli, rhaT, encoding the transporter (17, 39), and rhaR and rhaS, governing expression (40, 41), have been sequenced and extensively analyzed. In this species, another methylpentose, L-fucose, is metabolized by a parallel metabolic pathway integrated by a set of specific enzymes encoded by the fuc gene cluster, which has been located at 60.2 min (23) and completely sequenced (11,25). In Salmonella typhimurium LT2, rhaB, for rhamnulose kinase; rhaC2, one of the regulatory genes (31); and rhaT, encoding the transporter (39) have also been sequenced.Here we present a sequence analysis of three structural genes of the rhamnose pathway, some experiments involving their expression, and a comparison with the corresponding gene sequences of the L-fucose system. MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. Cells were grown aerobically as described previously (7) on LB or minimal medium. For growth on minimal medium, L-rhamnose, glucose, or L-fucose was added to 0.2%. When indicated, X-Gal (5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside) was added to 40 ,ug/ml. Ampicillin was used at 100 p,g/ml, kanamycin was used at 30 ,ug/ml, and streptomycin was used at 25 ,ug/ml. For primer extension analysis, the * Corresponding author. strains were grown in M10 medium (33) contain...