Annie Kolb

Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli.

Can a transcriptional activator known to bend DNA be functionally replaced by a sequence‐directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its ‐35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements. Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts. Some of these hybrid promoters turned out to be more efficient than the CRP activated wild‐type gal promoter in vivo. Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only. Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression. No significant stimulation of transcription could be detected in vitro. Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site. This partial analogy suggests that bending induced by the cAMP‐CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation.

Stringent spacing requirements for transcription activation by CRP.

The cyclic AMP receptor protein-cAMP complex (CRP-cAMP) binds at a variety of distances upstream of several E. coli promoters and activates transcription. We have constructed a model system in which a consensus CRP binding site is placed at different distances upstream of the melR promoter. CRP-cAMP activates transcription from melR when bound at a number of positions, all of which lie on the same face of the DNA helix. The two distances at which transcription is strongly activated correspond exactly to those at which CRP-cAMP binds upstream of the well-studied galP1 and lac promoters. Footprinting of the synthetic promoters reveals that RNA polymerase makes identical contacts with their -10 regions even though CRP-cAMP binds at a different distance in each case. Kinetic analysis in vitro indicates that CRP-cAMP activates transcription from these promoters in similar but distinct ways. A model is proposed to explain this two-position activation.

The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters

The AsiA protein, encoded by bacteriophage T4, inhibits Eσ70‐dependent transcription at bacterial and early‐phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the −35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C‐terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of σ70, which recognizes the −35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary‐phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a −35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Eσ70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of σ70 with the −35 hexamer.

σ factor selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and Lrp transcription factors

osmY is a stationary phase‐induced and osmotically regulated gene in Escherichia coli that requires the stationary phase RNA polymerase (EσS) for in vivo expression. We show here that the major RNA polymerase, Eσ70, also transcribes osmY in vitro and, depending on genetic background, even in vivo. The cAMP receptor protein (CRP) bound to cAMP, the leucine‐responsive regulatory protein (Lrp) and the integration host factor (IHF) inhibit transcription initiation at the osmY promoter. The binding site for CRP is centred at −12.5 from the transcription start site, whereas Lrp covers the whole promoter region. The site for IHF maps in the −90 region. By mobility shift assay, permanganate reactivity and in vitro transcription experiments, we show that repression is much stronger with Eσ70 than with EσS holoenzyme. We conclude that CRP, Lrp and IHF inhibit open complex formation more efficiently with Eσ70 than with EσS. This different ability of the two holoenzymes to interact productively with promoters once assembled in complex nucleoprotein structures may be a crucial factor in generating σS selectivity in vivo.

Crl activates transcription initiation of RpoS-regulated genes involved in the multicellular behavior of Salmonella enterica serovar typhimurium

In Salmonella enterica serovar Typhimurium, the stationary-phase sigma factor sigma(S) (RpoS) is required for virulence, stress resistance, biofilm formation, and development of the rdar morphotype. This morphotype is a multicellular behavior characterized by expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The Crl protein of Escherichia coli interacts with sigma(S) and activates expression of sigma(S)-regulated genes, such as the csgBAC operon encoding the subunit of the curli proteins, by an unknown mechanism. Here, we showed using in vivo and in vitro experiments that the Crl protein of Salmonella serovar Typhimurium is required for development of a typical rdar morphotype and for maximal expression of the csgD, csgB, adrA, and bcsA genes, which are involved in curli and cellulose biosynthesis. In vitro transcription assays and potassium permanganate reactivity experiments with purified His(6)-Crl showed that Crl directly activated sigma(S)-dependent transcription initiation at the csgD and adrA promoters. We observed no effect of Crl on sigma(70)-dependent transcription. Crl protein levels increased during the late exponential and stationary growth phases in Luria-Beratani medium without NaCl at 28 degrees C. We obtained complementation of the crl mutation by increasing sigma(S) levels. This suggests that Crl has a major physiological impact at low concentrations of sigma(S).

The CAP Modulon

The catabolite gene activator protein (CAP) is a transcription factor found in Escherichia coli. It was originally identified as a gene where disruptions suppressed the expression of the lactose (lac) operon, but it was rapidly realized that it had a role at a large number of other promoters. The story of the discovery of CAP is fascinating and has been told many times (e.g., by Pastan and Adhya,1 Ullmann and Danchin2). In the mid-sixties it had been shown that glucose repression of lac expression could be countered by the inclusion of cyclic AMP (cAMP) in the growth media. In the late sixties, mutants at two unlinked loci that suppressed lac expression were identified: the effects of the mutants at just one of the loci (at map position 85 minutes) could be suppressed by added cAMP. The simplest explanation was that the 85 minute mutations identified the gene encoding adenyl cyclase (cya), while the other locus, at 73 minutes, mapped a cAMP receptor. This triggered a rush to purify this receptor, and by the early seventies, simple in vitro systems could be used to show cAMP-depen-dent lac transcription. The rush also led to some confusion in nomenclature with the receptor being variously known as CRP (cyclic AMP receptor protein), CAP or CGA (catabolite gene activator protein).

DNA supercoiling contributes to disconnect σS accumulation from σS‐dependent transcription in Escherichia coli

The σS subunit of RNA polymerase is a key regulator of Escherichia coli transcription in stress conditions. σS accumulates in cells subjected to stresses such as an osmotic upshift or the entry into stationary phase. We show here that, at elevated osmolarity, σS accumulates long before the beginning of the σS‐dependent induction of osmEp, one of its target promoters. A combination of in vivo and in vitro evidence indicates that a high level of DNA negative supercoiling inhibits transcription by EσS. The variations in superhelical densities occurring as a function of growth conditions can modulate transcription of a subset of σS targets and thereby contribute to the temporal disconnection between the accumulation of σS and σS‐driven transcription. We propose that, in stress conditions leading to the accumulation of σS without lowering the growth rate, the level of DNA supercoiling acts as a checkpoint that delays the shift from the major (Eσ70) to the general stress (EσS) transcriptional machinery, retarding the induction of a subset of the σS regulon until the conditions become unfavourable enough to cause entry into stationary phase.

Involvement of differential efficiency of transcription by esigmas and esigma70 RNA polymerase holoenzymes in growth phase regulation of the Escherichia coli osmE promoter.

Transcription of the gene osmE of Escherichia coli is inducible by elevated osmotic pressure and during the decelerating phase of growth. osmE expression is directed by a single promoter, osmEp. Decelerating phase induction of osmEp is dependent on the σs (RpoS) factor, whereas its osmotic induction is independent of σs. Purified Eσs and Eσ70 were both able to transcribe osmEpin vitro on supercoiled templates. In the presence of rpoD800, a mutation resulting in a thermosensitive σ70 factor, a shift to non‐permissive temperature abolished induction of osmEp after an osmotic shock during exponential phase, but did not affect the decelerating phase induction. Point mutations affecting osmEp activity were isolated. Down‐promoter mutations decreased transcription in both the presence and the absence of σs, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the osmE promoter. Three up‐promoter mutations brought osmEp closer to the consensus of Eσ70‐dependent promoters. The two variant promoters exhibiting the highest efficiency became essentially independent of σsin vivo. Our data suggest that Eσs transcribes wild‐type osmEp with a higher efficiency than Eσ70. A model in which an intrinsic differential recognition contributes to growth phase‐dependent regulation is proposed. Generalization of this model to other σs‐dependent promoters is discussed.

Assays for transcription factor activity.

Transcription factors interact at promoters to modulate the transcription of genes. This chapter describes three in vitro methods that can be used to monitor their activity: transcript assays, abortive initiation assays, and potassium permanganate footprinting. These techniques have been developed using bacterial systems, and can be used to study the kinetics of transcription initiation, and hence to unravel regulatory mechanisms.

Upstream curved sequences influence the initiation of transcription at the Escherichia coli galactose operon

The two overlapping promoters that control mRNA synthesis at the galactose operon contain three phased stretches of adenine residues, located around positions -84.5, -74 and -63, with respect ot the start of the P1 promoter. As a result, the corresponding DNA sequence is bent, an anomaly that is relieved by the addition of small concentrations of drugs like distamycin A or netropsin. By abortive initiation assays performed on several DNA fragments derived from the wild-type promoter or from various mutants we show that the curved sequence increases the strength of the P1 promoter. In the absence of cyclic AMP (cAMP) and of the corresponding receptor protein (CRP), the upstream curved sequences enhance the rate of isomerization from the closed to the open complex at P1. This effect is abolished when distamycin A is bound in the bent region. In the presence of cAMP-CRP, a more drastic change is observed: activation of the gal P1 promoter takes place at a different formal step, depending whether the upstream curved sequence is present or not (enhancement of the rate of conversion from a closed to an open complex instead of an increase in the affinity of the enzyme during closed complex formation). These data, together with previous results obtained with other mutants of the gal control region, suggest that several closed complexes corresponding to different nucleoprotein arrangements are formed during open complex formation at gal P1, in the presence of CRP.