Regulation of the Escherichia coli rrnB P2 promoter

The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.     

Promoter recognition and promoter strength in the Escherichia coli system.

The strength of Escherichia coli promoters in vivo as well as the rates of association between RNA polymerase and promoter sequences differ by more than an order of magnitude. Since efficient promoter recognition and rapid binding of the enzyme might be a prerequisite for exceptional promoter strength we have determined the forward rate constants kon (as well as koff) for nine promoters including PL, PA1, and PN25 from phages lambda, T7, and T5, respectively as well as Pbla and PlacUV5 from E. coli. The second order forward rate constants span a 30-fold range from 1 X 10(7) M-1 s-1 for Pbla and PL up to 2.9 X 10(8) M-1 S-1 for PN25. Little correlation between 'promoter recognition' as defined by the rate of complex formation of a promoter sequence with RNA polymerase and its strength in vivo as defined by the rate of RNA synthesis has been found. This adds to the evidence that the complex functional pathway encoded in a promoter sequence can be limited at various levels and that promoter strength in vivo is the result of an optimization process involving more than just one functional parameter.     

In vivo regulation of the Escherichia coli araC promoter.

The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose. This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions. In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible. Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants. Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.     

The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor

We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor. By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor. We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor. The alpha amino group, however, has a small negative contribution to the affinity of L-tryptophan for the aporepressor which may be associated with its essential role in operator-specific binding.     

The structural basis of receptor-binding by Escherichia coli associated with diarrhea and septicemia

GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7A resolution in the presence of the receptor sugar N-acetyl-D-glucosamine. The overall fold is a beta-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.