Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of differences in gene expression due to small adaptive mutations in RNA polymerase B' subunit (rpoC)

ABSTRACT: Studies of the RNA polymerase-binding molecule ppGpp in bacteria and plants have shown that changes to the kinetics of the RNA polymerase can have dramatic biological effects in the short-term as a stress response. Here we describe the reprogramming of the kinetic parameters of the RNAP through mutations arising during laboratory adaptive evolution of Escherichia coli in minimal media. The mutations cause a 10- to 30-fold decrease in open complex stability at a ribosomal promoter and approximately a 10-fold decrease in transcriptional pausing in the his operon. The kinetic changes coincide with large scale transcriptional changes, including strong downregulation of motility, acid-resistance, fimbria, and curlin genes which are observed in site-directed mutants containing the RNA polymerase mutations as well as the evolved strains harboring the mutations. Site-directed mutants also grow 60% faster than the parent strain and convert the carbon-source 15% to 35% more efficiently to biomass. The results show that long-term adjustment of the kinetic parameters of RNA polymerase through mutation can be important for adaptation to a condition. Mutations in the RNA polymerase beta prime subunit (rpoC) were discovered in E. coli following adaptation to continual logarithmic growth (OD <= 0.3) in glycerol M9 minimal media at 30 C. We used site-directed mutagenesis to make strains of E. coli isogenic to wild-type except for single adaptive rpoC mutations. We found they increase growth rate in this condition by 60%. In order to understand how the mutations affect gene expression in this condition, we extracted total mRNA from the strains, which had been growing in the adaptive evolution condition, at OD=0.3. There were three RNAP mutants and the wild-type (E. coli K-12 MG1655). Each strain had three flasks from which RNA was extracted (three biological replicates). There were no technical replicates. The mRNA was synthesized into cDNA, labeled, and hybridized to an Affymetrix E. coli 2.0 GeneChip.

ORGANISM(S): Escherichia coli

SUBMITTER: Bernhard Palsson

PROVIDER: E-GEOD-15500 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Analysis of differences in gene expression due to small adaptive mutations in RNA polymerase B' subunit (rpoC)

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