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Sigma32 over-expression in E. coli

ABSTRACT: This study examined the genes under the control of sigma32 in E. coli by moderate induction of a plasmid-borne rpoH gene under defined steady-state growth condition. Samples were taken from culture at mid log phase (OD=0.2) before or 5 minutes, 10 minutes or 15 minutes after induction. Samples were then RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChip E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com). Meanwhile, we measured the protein level of sigma32 during the time course. The changes of some sigma32 –dependent genes were also determined in a DnaK null mutant to examine the anti- sigma32 function of DnaK. Keywords = sigma factor Keywords = regulon Keywords = microarray Keywords = Western blot Keywords = E. coli Keywords = anti-sigma factor Keywords: time-course

ORGANISM(S): Escherichia coli K-12 Escherichia coli

PROVIDER: GSE2140 | GEO | 2007/08/01

SECONDARY ACCESSION(S): PRJNA91269

REPOSITORIES: GEO

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Project description:When performed at single bp resolution, the genome-wide location, occupancy level, and structural organization of DNA binding proteins provides mechanistic insights into genome regulation. Here we use ChIP-exo to provide the first high resolution view of the epigenomic organization of the E. coli transcription machinery and nucleoid structural proteins when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We suggest that indirect readout of DNA shape at the flanks of cognate motifs provide major contributions to site specificity at promoter positions -35/-24 and -10/-12. We examined the site specificity of three sigma factors (RpoD/70, RpoH/32, and RpoN/54), RNA polymerase (RNAP or RpoA, B, C) and two nucleoid proteins (Fis and IHF). Our results confirm and refine reports that RpoD binds most annotated promoters, whereas RpoH and RpoN bind a much smaller subset, each through their cognate motifs. However, only upon heat shock does RpoH becomes active for RNAP recruitment. In contrast, upon heat shock RpoD remains active at its cognate promoters (including at heat shock genes), whereas RpoN remains inactive at its cognate promoters. RpoN binds ~1,000 non-annotated RpoN motifs, which may reflect a large number of condition-specific transcription units. Occupancy patterns of sigma factors and RNAP suggest a common promoter recruitment mechanism that differs from the long-standing views that sigma and RNAP are co-recruited as a complex, and also simultaneously dissociate from promoters. Our findings suggest that sigma factors are recruited and/or maintained at most promoters via an RNAP-independent mechanism. When RNAP arrives, it dwells for a relatively short time before clearing the promoter, leaving sigma behind. Taken together these findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape and redefine mechanistic steps in regulated promoter assembly in E. coli.

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Sigma32 over-expression in E. coli

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