Cassette mutagenesis implicates a helix-turn-helix motif in promoter recognition by the novel RNA polymerase Sigma factor σ54

Cassette mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.     

Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters

Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.     

m-xylene-responsive Pu-PnifH hybrid sigma54 promoters that overcome physiological control in Pseudomonas putida KT2442

The sequences surrounding the -12/-24 motif of the m-xylene-responsive sigma54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH sigma54 promoter variants known to have various degrees of efficacy and affinity for sigma54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the -12/-24 region of the wild-type PnifH and its higher sigma54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.     

Promoter selection by a bacterial enhancer-like activator element (BELE) in Escherichia coli

The Escherichia coli glnA gene promoter glnAp2 is activated by an element able to act bidirectionally and at variable distance over the DNA. We demonstrate here that this activating element does not influence another promoter, 82p, adjacent to it, from which a gene is transcribed in opposite direction to glnA. Thus, although it displays a great flexibility, this element can activate selectively. The unresponsive promoter and glnAp2 are recognized by RNA polymerases complexed to two different sigma factors. Therefore, we argue that promoter selection by this element is dependent upon distinguishing the proper sigma factor.