Deoxyribonucleic acid-binding studies on the hut repressor and mutant forms of the hut repressor of Salmonella typhimurium

Abstract

In Salmonella typhimurium the genes coding for the enzymes of histidine utilization (hut) are clustered in two adjacent operons, hutMIGC and hut(P,R,Q)UH. A single repressor, the product of the C gene, regulates both operons by binding at two operator sites, one near M and one in (P,R,Q). The deoxyribonucleic acid (DNA)-binding activity of the repressor was measured using DNAs containing separate operators. The repressor had greater activity when assayed using DNA containing the operator of the (P,R,Q)UH operon than when assayed using DNA containing the operator of the MIGC operon. The binding to either operator was absent in the presence of the inducer, urocanate. The DNA-binding activities were also determined for two super-repressors. The super-repressors had altered DNA-binding properties, although the self-regulated nature of the repressors complicated the analysis of the results. A purification procedure for the wild-type repressor is presented. The purified repressor was somewhat unstable, and additional experiments using it were not performed.In Salmonella typhimurium, the genes coding for the four enzymes of histidine utilization (hut) are clustered in two adjacent operons, hutMIGC and hut(P,R,Q)UH (1,9,(15)(16)(17). A single repressor regulates both operons. The gene coding for the repressor, C, is itself a member of one of the operons (the MIGC operon), and the repressor thus regulates its own synthesis (5,15). There are two distinct promoter-operator regions, one in the MIGC operon near M, and one in the (P,R,Q)UH operon in (P,R,Q). Transcription is rightward from these two regions. Urocanate, the first degradation product of histidine and the inducer of the hut operons, prevents the binding of the repressor at either operator. (The hut regulatory mechanism is illustrated in Fig. 1.) M and (P,R,Q) are cis-acting control regions of the two operons (17). Mutations in M result in an increased level of 4-imidazolone-5-propionate amidohydrolase (the product of the I gene), N-formimino-L-glutamate formiminohydrolase) (FGA hydrolase) (the product of the G gene), and repressor in cells grown in the presence of inducer (16,17). No operator mutations have yet been found in the MIGC operon. (An operator undoubtedly does exist and, most probably, is located in the vicinity of M.) 1 Present address: Department ofBiochemistry, Stanford University, Stanford, Calif. 94305.(P,R,Q) represents a promoter-operator complex (1,9,17). Mutations in P result in a greatly reduced level of histidase (the product of theH gene) and urocanase (the product of the U gene) in cells grown in the presence of inducer. Mutations in R render the synthesis of histidase and urocanase insensitive to catabolite repression. Mutations in Q render the synthesis of histidase and urocanase partially constitutive and insensitive to catabolite repression.There are two indications that the repressor has different affinities for the two operators, the affinity for the operator of the MIGC operon being less than the affinity for the operator of the (P,...

Transcription of the hut operons of Salmonella typhimurium

“…RNA preparations devoid of bacterial RNA homologous to Xphut36 DNA (a) were from strain NE547 growing on SNH medium. the hybridization reaction mixture and replaced with fresh filters for a second round of hybridization (experiments [3][4][5][6]. Examination of the data shows that: (i) a negligible amount of hybrid was formed with DNA, regardless of the source of the RNA preparation used in the reaction; (ii) essentially no hybrid was formed with Xphut36 DNA using RNA preparations from strain NE547 (gal-hut-bioA); (iii) approximately 95% of the RNA homologous to Xphut36 DNA in preparations from strain NE449 was removed during the first round of hybridization (cf.…”

“…The hutI and hutG genes, which are responsible for the third and fourth enzymes of the pathway, are located in the "left-hand operon" along with the hutC gene (13,14). The hutC gene product is a repressor that binds to two separate operator sites in the hut region (4). The enzymes of the two hut operons exhibit quite different levels of inducibility and are differentially sensitive to catabolite repression (2,10,(12)(13)(14).…”