Stephen J. W. Busby

The regulation of bacterial transcription initiation

Bacteria use their genetic material with great effectiveness to make the right products in the correct amounts at the appropriate time. Studying bacterial transcription initiation in Escherichia coli has served as a model for understanding transcriptional control throughout all kingdoms of life. Every step in the pathway between gene and function is exploited to exercise this control, but for reasons of economy, it is plain that the key step to regulate is the initiation of RNA-transcript formation.

Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome

The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.

Region 2.5 of the Escherichia coli RNA polymerase σ70 subunit is responsible for the recognition of the ‘extended −10’ motif at promoters

At some bacterial promoters, a 5′‐TG‐3′ sequence element, located one base upstream of the −10 hexamer element, provides an essential motif necessary for transcription initiation. We have identified a mutant of the Escherichia coli RNA polymerase σ70 subunit that has an altered preference for base sequences in this ‘extended −10’ region. We show that this mutant σ70 subunit substantially increases transcription from promoters bearing 5′‐TC‐3′ or 5′‐TT‐3′ instead of a 5′‐TG‐3′ motif, located one base upstream of the −10 hexamer. The mutant results from a single base pair substitution in the rpoD gene that causes a Glu to Gly change at position 458 of σ70. This substitution identifies a functional region in σ70 that is immediately adjacent to the well‐characterized region 2.4 (positions 434–453, previously shown to contact the −10 hexamer). From these results, we conclude that this region (which we name region 2.5) is involved in contacting the 5′‐TG‐3′ motif found at some bacterial promoters: thus, extended −10 regions are recognized by an extended region 2 of the RNA polymerase σ70 subunit.

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.

Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression

Bacterial nucleoid-associated proteins play a key role in the organisation, replication, segregation, repair and expression of bacterial chromosomes. Here, we review some recent progress in our understanding of the effects of these proteins on DNA and their biological role, focussing mainly on Escherichia coli and its chromosome. Certain nucleoid-associated proteins also regulate transcription initiation at specific promoters, and work in concert with dedicated transcription factors to regulate gene expression in response to growth phase and environmental change. Some specific examples, involving the E. coli IHF and Fis proteins, that illustrate new principles, are described in detail.

The bacterial LexA transcriptional repressor

Abstract.Bacteria respond to DNA damage by mounting a coordinated cellular response, governed by the RecA and LexA proteins. In Escherichia coli, RecA stimulates cleavage of the LexA repressor, inducing more than 40 genes that comprise the SOS global regulatory network. The SOS response is widespread among bacteria and exhibits considerable variation in its composition and regulation. In some well-characterised pathogens, induction of the SOS response modulates the evolution and dissemination of drug resistance, as well as synthesis, secretion and dissemination of the virulence. In this review, we discuss the structure of LexA protein, particularly with respect to distinct conformations that enable repression of SOS genes via specific DNA binding or repressor cleavage during the response to DNA damage. These may provide new starting points in the battle against the emergence of bacterial pathogens and the spread of drug resistance among them.

Regulation of Acetyl Coenzyme A Synthetase in Escherichia coli

Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.

Extensive functional overlap between σ factors in Escherichia coli

Bacterial core RNA polymerase (RNAP) must associate with a σ factor to recognize promoter sequences. Escherichia coli encodes seven σ factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of σ32, the heat-shock σ factor, and estimate that there are 120–150 σ32 promoters in total. Unexpectedly, 25% of these σ32 targets are located within coding regions, suggesting novel regulatory roles for σ32. The majority of σ32 promoter targets overlap with those of σ70, the housekeeping σ factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPσ70 and RNAPσ32 initiating transcription in vitro with similar efficiency and from identical positions. σE-regulated promoters also overlap extensively with those for σ70. These results suggest that extensive functional overlap between σ factors is an important phenomenon.

A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria

The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome C552 of Escherichia coli K‐12 have been reported. We have now established that nrfA is the first gene in a seven‐gene operon, designated the nrf operon, at least five of which are essential for formate‐dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium‐independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR‐dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter, in contrast, expression of the gltP‐lac fusion was FNR‐independent.

Positive activation of gene expression

Most bacterial transcription activators function by making direct contact with RNA polymerase at target promoters. Some activators contact the carboxy-terminal domain of the RNA polymerase alpha subunit, some contact region 4 of the sigma70 subunit, whilst others interact with other contact sites. A number of activators are ambidextrous and can, apparently simultaneously, contact more than one target site on RNA polymerase. Expression from many promoters is co-dependent on two or more activators. There are several different mechanisms for coupling promoter activity to more than one activator: in one such mechanism, the different activators make independent contacts with different target sites on RNA polymerase.