Ss-LrpB, a novel Lrp-like regulator of Sulfolobus solfataricus P2, binds cooperatively to three conserved targets in its own control region.

Ss-LrpB, a novel Lrp-like DNA-binding protein from the hyperthermophilic crenarchaeon Sulfolobus solfataricus, was shown to bind cooperatively to three regularly spaced targets in its own control region, with as consensus the 15 bp palindrome 5'-TTGYAW WWWWTRCAA-3'. Binding to the border sites occurred with high affinity; the target in the middle proved to be a low affinity site which is stably bound only when both flanking sites are occupied. Ss-LrpB contacts two major groove segments and the intervening minor groove of each site, all aligned on one face of the helix. The operator shows intrinsic bending and is increasingly deformed upon binding of Ss-LrpB to one, two and three targets. Complex formation relies therefore on DNA conformability, protein-DNA and protein-protein contacts. Mobility-shift assays and in gel footprinting indicate that Ss-LrpB and the transcription factors TATA-box binding protein (TBP) and transcription factor B (TFB) can bind simultaneously to the control region. Based on these findings we present a model for the construction of the higher order nucleoprotein complexes and a hypothesis for the autoregulatory process. The latter is based on the concentration-dependent formation of distinct complexes exhibiting different stoichiometries and conformations, which could positively and negatively affect promoter activity.

Purine and pyrimidine-specific repression of the Escherichia coli carAB operon are functionally and structurally coupled.

Transcription of the carAB operon encoding the sole carbamoylphosphate synthetase of Escherichia coli proceeds from a tandem pair of promoters. P2, downstream, is repressed by arginine and the ArgR protein, whereas P1 is submitted to pyrimidine-specific regulation and as shown here to purine-specific control exerted by binding of the PurR protein to a PUR box sequence centered around nucleotide -128.5 with respect to the start of P1 transcription. In vivo analyses of the effects of trans and cis-acting mutations on the regulatory responses and single round in vitro transcription assays indicated that ligand-bound PurR is by itself unable to inhibit P1 promoter activity. To exert its effect PurR relies on the elaborated nucleoprotein complex that governs P1 activity in a pyrimidine-specific manner. Thus we reveal the existence of an unprecedented functional and structural coupling between the modulation of P1 activity by purine and pyrimidine residues that appears to result from the unique position of the PUR box in the carAB control region, far upstream of the promoter. Missing contact and premethylation binding interference studies revealed the importance of base-specific groups and of structural aspects of the PUR box sequence in complex formation. Permutation assays indicated that the overall PurR-induced bending of the carAB control region is slightly less pronounced than that of the purF operator. The PUR boxes of the carAB operon of E.coli and Salmonella typhimurium are unique in that they have a guanine residue at position eight. Interestingly, guanine at this position has been proposed to be extremely unfavorable on the basis of modeling and binding studies, as its exocyclic amino group would enter into a steric clash with the side-chain of lysine 55. To analyze the effect of guanine at position eight in the upstream half-site of the carAB operator we constructed the adenine derivative and assayed in vivo repressibility of P1 promoter activity and in vitroPurR binding to the mutant operator, and constructed a molecular model for the unusual lysine 55-guanine 8 interaction.

Genes of de novo pyrimidine biosynthesis from the hyperthermoacidophilic crenarchaeote Sulfolobus acidocaldarius: novel organization in a bipolar operon.

Sequencing a 8,519-bp segment of the Sulfolobus acidocaldarius genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers, pyrEF-orf8 and pyrBI-orf1-pyrCD-orf2-orf3-orf4, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of pyr genes that also occurs in the recently determined genome sequences of Sulfolobus solfataricus P2 and Sulfolobus tokodaii strain 7; the configuration appears therefore characteristic of Sulfolobus. The quasi-leaderless pyrE and pyrB genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3' end of 16S rRNA. The polycistronic nature of the pyr messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling. pyrB transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate pyrB transcription in bacteria. In contrast, the pyrE-pyrB promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).