Published: August 2008
Authors: Grainger DC, Busby SJ.
Abstract:
A major goal in the study of gene regulation is to untangle the transcription-regulatory networks of Escherichia coli and other 'simple' organisms. To do this we must catalogue the binding sites of all transcription factors. ChIP (chromatin immunoprecipitation), combined with DNA microarray analysis, is a powerful tool that permits global patterns of DNA binding to be measured. Here, we discuss the benefits of this approach and the application of this technique to bacterial systems.
Published: 8 May 2009
Authors: Sala C, Grainger DC, Cole ST.
Abstract:
Understanding host-microbe interactions has been greatly enhanced by our broadening knowledge of the regulatory mechanisms at the heart of pathogenesis. The "transcriptomics" approach of measuring global gene expression has identified genes involved in bacterial pathogenesis. More recently, chromatin immunoprecipitation (ChIP) and hybridization to microarrays (chIP-on-chip) has emerged as a complementary tool that permits protein-DNA interactions to be studied in vivo. Thus, chIP-on-chip can be used to map the binding sites of transcription factors, thereby teasing apart gene regulatory networks. In this Review, we discuss the ChIP-on-chip technique and focus on its application to the study of host-pathogen interactions.
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Published: October 2010
Authors: Grainger DC, Lee DJ, Busby SJ.
Abstract:
Transcription factors and sigma factors play a major role in bacterial gene regulation by guiding the distribution of RNA polymerase between the promoters of different transcription units in response to changes in the environment. For 40 years Escherichia coli K-12 has been the paradigm for investigating this regulation and most studies have focused on small numbers of promoters studied by a combination of genetics and biochemistry. Since the first complete sequence for a bacterial genome was reported, the emphasis has switched to studying transcription on a global scale, with transcriptomics and bioinformatics becoming the methods of choice. Here we discuss two complementary direct experimental methods for studying transcription factors and sigma factors and we outline their potential use in rapidly establishing the regulatory networks in newly sequenced bacteria.
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Published: September 2010
Authors: Sánchez-Romero MA, Busby SJ, Dyer NP, Ott S, Millard AD, Grainger DC.
Abstract:
The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding factor and chaperone that is expressed at high levels as cells enter stationary phase. Using a combination of genetics, biochemistry, structural modelling and single-molecule atomic force microscopy we have examined dimerization of, and DNA binding by, CbpA. Our data show that CbpA dimerization is driven by a hydrophobic surface comprising amino acid side chains W287 and L290 located on the same side of an ? helix close to the C-terminus of CbpA. Derivatives of CbpA that are unable to dimerize are also unable to bind DNA. Free in solution, CbpA can exist as either a monomer or dimer. However, when bound to DNA, CbpA forms large aggregates that can protect DNA from degradation by nucleases. These CbpA-DNA aggregates are similar in morphology to protein-DNA complexes formed by the DNA-binding protein from starved cells (Dps), the only other stationary phase-specific nucleoid protein. Conversely, protein-DNA complexes formed by Fis, the major growth phase nucleoid protein, have a markedly different appearance.
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Published: 18 May 2010
Authors: Sánchez-Romero MA, Busby SJ, Dyer NP, Ott S, Millard AD, Grainger DC.
Abstract:
The bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coli K-12, the single replication origin oriC is a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequesters oriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entire Escherichia coli K-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in the ter macrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.
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Published: December 2010
Authors: Browning DF, Grainger DC, Busby SJ.
Abstract:
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.
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Published: March 2011
Authors: Yamamoto K, Ishihama A, Busby SJ, Grainger DC.
Abstract:
Escherichia coli MntR protein is the Mn(2+)-responsive transcriptional repressor of the MntH manganese transporter. We have used chromatin immunoprecipitation to determine the distribution of Mn(2+)-MntR across the entire E. coli chromosome in vivo, and we report that MntR binds to only four targets, adjacent to the mntH, mntR, yebN, and dps genes. Unexpectedly, we found that dps expression is directly repressed by Mn(2+)-MntR.
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Published: July 2011
Authors: Singh SS, Typas A, Hengge R, Grainger DC.
Abstract:
In bacteria, promoter identification by RNA polymerase is mediated by a dissociable ? factor. The housekeeping ?(70) factor of Escherichia coli recognizes two well characterized DNA sequence elements, known as the '-10' and '-35' hexamers. These elements are separated by 'spacer' DNA, the sequence of which is generally considered unimportant. Here, we use a combination of bioinformatics, genetics and biochemistry to show that ?(70) can sense the sequence and conformation of the promoter spacer region. Our data illustrate how alterations in spacer region sequence can increase promoter activity. This stimulatory effect requires ?(70) side chain R451, which is located in close proximity to the non-template strand at promoter position -18. Conversely, R451 is not required to mediate transcriptional stimulation by improvement of the -10 element. Mutation of ?(70) residue R451, which is highly conserved, results in reduced growth rate, consistent with a central role in promoter recognition.
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Published: 12 August 2011
Authors: Chintakayala K, Grainger DC.
Abstract:
Hsp40-like co-chaperones are ubiquitous enzymes that stimulate the protein refolding activity of Hsp70 family chaperones. They are widespread in prokaryotic and eukaryotic systems. In bacteria, the best characterized co-chaperone is the Escherichia coli DnaJ protein. Many ?-proteobacteria encode a functional homologue of DnaJ, known as CbpA, which is expressed in response to starvation and environmental stress. The activity of CbpA is regulated by the "modulator" protein CbpM. Here, we have used a combination of genetics and biochemistry to identify the co-chaperone contact determinant of CbpM. We show that the nature of the interaction is conserved in enterobacteria.
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