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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.

RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response to L-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether 70 might be an activation target for RhaS. We found that 70 K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged 70 residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and 70 substitutions and found that RhaS D241 and 70 R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that 70 R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact 70 . More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with 70 may be conserved.

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...

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The RhaS and RhaR proteins are transcription activators that respond to the availability of L-rhamnose and activate transcription of the operons in the Escherichia coli L-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the L-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the L-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the ؊35 hexamers of the promoters they regulate, suggesting they may contact the 70 subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and 70 residue R599. In the present study, we first identified two positively charged amino acids in 70 , K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and 70 R599. Finally, previous results from our lab separately showed that RhaS D250A and 70 K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and 70 .Transcription activation in Escherichia coli often involves the interaction of a DNA-binding activator protein with one of the subunits of RNA polymerase (RNAP), most often the sigma () or alpha (␣) subunit. Transcription activators that bind immediately upstream and adjacent to RNAP, in some cases overlapping the Ϫ35 promoter hexamer, may interact with the C-terminal domain (domain 4) of the subunit of RNAP (8,27). The cI protein of bacteriophage is required for the establishment and maintenance of lysogeny and is perhaps the best-characterized example of a transcription activator that contacts 70 . The cI protein activates transcription of the P RM promoter when bound at the O R 2 operator site, which overlaps the P RM Ϫ35 hexamer by 2 bp (30). Current evidence suggests that 70 residues R588, K593, and R596 are required for activation by cI (23,26,35). Genetic and molecular modeling studies, as well as the recent structure of a ternary cI-domain 4-DNA complex, indicate that cI D38 contacts both 70 K593 ( A K418) and R596 ( A R421) and cI E34 contacts 70 R588 ( A R413) (8,19,26,35). Prior to the identification of the ternary complex structure, a molecular model of the interaction indicated that 70 K593 ( A K418) contacts DNA but was not positioned to contact cI (6,8,35). However, the ternary structure showed that the A residue that aligns with 70 K593 has moved away from the DNA (relative to the model of the interaction) and instead makes a proteinprotein contact with cI D38 (19).There is also evidence that activation by several tran...

During anaerobic growth, nitrate induces synthesis of the anaerobic respiratory enzymes formate dehydrogenase-N and nitrate reductase. This induction is mediated by a transcription activator, the narL gene product. The narX gene product may be involved in sensing nitrate and phosphorylating NARL. We isolated narX mutants, designated narX*, that caused nitrate-independent expression of the formate dehydrogenase-N and nitrate reductase structural genes. We used lambda narX specialized transducing phage to genetically analyze these lesions in single copy. Two previously isolated narX* mutations, narX32 and narX71, were also constructed by site-specific mutagenesis. We found that each of these alleles caused nitrate-independent synthesis of formate dehydrogenase-N and nitrate reductase, and each was recessive to narX+. The narX* mutations lie in a region of similarity with the methyl-accepting chemotaxis protein Tsr. We suggest that the narX* proteins have lost a transmembrane signalling function such that phosphoprotein phosphatase activity is reduced relative to protein kinase activity.