Stephen D. Minchin

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.

A DNA expression array to detect toxic stress response in European flounder (Platichthys flesus).

As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress-related and other genes from European flounder cDNA and genomic DNA. Additionally, 22 fragments were obtained by suppressive subtractive hybridisation (SSH). These fragments were cloned and sequenced, then, with additional control genes, used to create a cDNA microarray for flounder. After optimisation of the arraying process, hepatic mRNA was isolated from flounder caught in the polluted Tyne and relatively unpolluted Alde estuaries. Fluorescent cDNA probes were synthesised from the mRNA and used in dual-colour hybridisations to the microarray. A number of transcripts were differentially expressed between Tyne and Alde female flounder but these changes were not significant, due to high inter-individual variation. However, in comparisons between Tyne and Alde male flounder, 11 transcripts were found to significantly differ in expression (P<0.05). Seven transcripts were more highly expressed in the Tyne male fish (CYP1A, UDPGT, alpha-2HS-glycoprotein, dihydropyrimidine dehydrogenase, Cu/Zn SOD, aldehyde dehydrogenase and paraoxonase). Four transcripts (Elongation factor 1 (EF1), EF2, Int-6 and complement component C3) were found to be significantly less abundant in the Tyne male fish. Selected genes were assayed by real-time PCR, then normalised to alpha-tubulin. These assays confirmed the significance of the array results for CYP1A, UDPGT and EF1, but not for Cu/Zn SOD. This study provides a link between traditional single-gene biomarker studies and the emerging field of eco-toxicogenomics, demonstrating the utility of microarray studies on environmentally sampled, non-model organisms.

Activating Transcription in Bacteria

Bacteria use a variety of mechanisms to direct RNA polymerase to specific promoters in order to activate transcription in response to growth signals or environmental cues. Activation can be due to factors that interact at specific promoters, thereby increasing transcription directed by these promoters. We examine the range of architectures found at activator-dependent promoters and outline the mechanisms by which input from different factors is integrated. Alternatively, activation can be due to factors that interact with RNA polymerase and change its preferences for target promoters. We summarize the different mechanistic options for activation that are focused directly on RNA polymerase.

Extended —10 Promoters

E. coli RNA polymerase (RNAP) is a multisubunit enzyme with a molecular mass of nearly half a million. The major form of RNAP in cells consists of core enzyme (subunit composition ββ′α2) in complex with the σ70 factor. σ70 is 613 amino acids in length: sequence comparisons show that it shares four regions of amino acid sequence similarity with other σ factors (regions 1, 2, 3 and 4: Fig. 1; Gross et al. 1992). It has long been known that RNAP containing σ70, is competent to initiate transcription at many promoters in the absence of any activator protein and that the σ70 subunit is essential for recognition of these promoters.

Substitutions in the Escherichia coli RNA polymerase σ70 factor that affect recognition of extended −10 elements at promoters

Previous work has shown that the base sequence of the DNA segment immediately upstream of the −10 hexamer at bacterial promoters (the extended −10 element) can make a significant contribution to promoter strength. Guided by recently published structural information, we used alanine scanning and suppression mutagenesis of Region 2.4 and Region 3.0 of the Escherichia coli RNA polymerase σ70 subunit to identify amino acid sidechains that play a role in recognition of this element. Our study shows that changes in these regions of the σ70 subunit can affect the recognition of different extended −10 element sequences.

Analysis of mechanisms of activation and repression at bacterial promoters.

In bacteria, the expression of transcription units is controlled by regulatory regions, that contain one or more promoters and binding sites for regulatory proteins that activate or repress expression in response to different signals. In this chapter, we explain the diverse approaches that can be used to understand the mechanisms by which the different factors intervene, and how the effects are integrated. Bioinformatics, genetics and biochemistry must be combined to understand the organisation of regulatory regions and the mechanisms by which transcription initiation is controlled.

Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1+ and S1-), but only the S2- from of the S2 splice variant of the type I InsP3 receptor. PCR analysis was also used to identify InsP3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [3H]-InsP3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a Bmax of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The Kd for InsP3 binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 +/- 20 nM). InsP3-induced Ca2+ release (IICR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal response) of 0.5 +/- 0.03 microM, also similar to that seen for cerebellar microsomes (0.3 microM). Maximal IICR occurred at about 20 microM InsP3, with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s-1 at 30 microM InsP3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (approximately 1 s-1) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is approximately 8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP3 analogues also confirmed similar behaviour for testicular InsP3Rs and cerebellar InsP3Rs.

Positioning of region 4 of the Escherichia coli RNA polymerase sigma(70) subunit by a transcription activator.

A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase sigma(70) subunit, has been used to investigate the location of sigma(70) region 4 in different complexes at the galp(1) promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp(1) promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of sigma(70) region 4.

Unwinding of duplex DNA during transcription initiation at the Escherichia coli galactose operon overlapping promoters

We have used potassium permanganate as a probe to detect DNA duplex unwinding in vitro, in open complexes between E. coli RNA polymerase and DNA fragments carrying the E. coli galactose operon regulatory region. This zone contains 3 overlapping promoters which specify transcription initiation at 3 distinct startpoints. We have used mutant gal derivatives carrying different single point mutations, each of which allows initiation from only one of the 3 start sites. This has allowed us to compare duplex unwinding in open complexes at the 3 different promoters, and to show that the extent of the unwinding is similar in each case. Further, the pattern of DNA modification by potassium permanganate suggests a model for discrimination between the upper and lower strands. Finally, we show that DNA modification by potassium permanganate at the gal promoters is the same in vivo as in vitro.

Characterisation of the connexin32 promoter and changes in response element complexes in rat liver and hepatocytes during culture associated with oxidative stress

Hepatic gap junctional intercellular communication (GJIC), mediated principally by connexin 32, provides a mechanism for regulating multicellular activities between neighbouring cells. The control of Cx32 gene expression at the transcriptional level has been investigated in rat liver tissue and in primary rat hepatocytes during culture. Several response elements have been identified and characterised using the electrophoretic mobility shift assay. Nuclear protein extract prepared from rat primary hepatocytes cultured for 2 h gave a larger number of DNA-protein complexes than observed with extracts from liver in vivo, including complexes containing Sp1. In contrast, nuclear extracts prepared from primary rat hepatocytes cultured for 96 h, and subject to oxidative stress, gave altered DNA-protein complexes when compared to those from hepatocytes cultured for 2 h. These results indicate that culture conditions, known to cause a loss of connexin expression, can modulate the transcription of Cx32 in hepatocytes by affecting the regulatory trans/cis-interactions of redox-sensitive zinc finger proteins within the promoter.