PMID:19150431

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Citation

Mooney, RA, Davis, SE, Peters, JM, Rowland, JL, Ansari, AZ and Landick, R (2009) Regulator trafficking on bacterial transcription units in vivo. Mol. Cell 33:97-108

Abstract

The trafficking patterns of the bacterial regulators of transcript elongation sigma(70), rho, NusA, and NusG on genes in vivo and the explanation for promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters (rho first, then NusA, then NusG). However, the interactions of elongating complexes with these regulators did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from sigma(70) peaks in the direction of transcription and co-occurred with NusA and rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of rho did not increase RNAP levels within genes downstream from the RNAP peaks, suggesting the peaks are caused by a mechanism other than rho-dependent attenuation.

Links

PubMed PMC2747249 Online version:10.1016/j.molcel.2008.12.021

Keywords

Chromatin Immunoprecipitation; DNA-Directed RNA Polymerases/metabolism; Escherichia coli/genetics; Escherichia coli Proteins/metabolism; Genes, Bacterial; Models, Genetic; Promoter Regions, Genetic/genetics; Protein Binding; Protein Transport; Transcription, Genetic

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Brief Description

This paper uses ChIP-chip methods to investigate the occupancy of 5 proteins along the E. coli chromosome:

Main Points

  • There is not a one-to-one relationship between the (observed) occupancy of σ70 and RNAP.
  • Evidence sustaining the idea that NusA displaces σ70 during transcript elongation. (Previously reported in [1])
  • Elongating RNAPs do not exhibit transcription-unit specific variations in affinities for σ70, NusA, NusG or ρ. This suggests these proteins are general elongation regulators and not specific regulators (as in the case of rfaH.)
  • σ70 binds almost exclusively to the promoter regions.
  • NusG is associated with elongation complexes more slowly than NusA (on most transcription units.)
  • ρ binding is most evident between the σ70 and RNAP signals near the promoter. This suggests ρ may associate with the nascent transcripts soon after transcription initiation.

Methods and Calculations

High Quality TUs
109 "high-quality" transciption units were identified in which overlap from adjacent transcription units did not obscure the patterns. (Fig 1D & Table S1)
RNA Polymerase Control
170 regions of the genome (greater than 1kb in length) were identified as similar in transcript level to the cryptic gene bglB. This gave a non-specific background - see Fig 1B & 1C.
Apparent Occupancy
The Occapp was defined by linearly scaling the signals for each protein between 0 (basal level of RNAP) and 1 (the average of the 10 3-probe clusters with highest average value).
  • IP samples were compared to control samples of input DNA (recovered from cell extract immediately prior to IP.)
  • The microarray contains two copies of 187,204 Tm-matched ≥45bp oligos.
    • This tiled the chromosome with an average of 24.5bp separation.

EcoliWiki data manipulation

In order to show the results of this paper across the entire genome, we first downloaded the data-sets (GSE13938) from the Gene Expression Omnibus (GEO).

Tracks for the PortEco GBrowse were added for

[Category:GBrowse]

Annotations

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Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status
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Data

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Strain/Organism Assay Analyte Variable Conditions Notes Links

MG1655

ChIP-chip

NusA

MG1655

ChIP-chip

NusG

MG1655

ChIP-chip

Rho

MG1655

ChIP-chip

RNAP

MG1655

ChIP-chip

RpoD (Sigma70)

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EcoliWiki Links

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See also

References

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  1. ↑ Greenblatt, J & Li, J (1981) Interaction of the sigma factor and the nusA gene protein of E. coli with RNA polymerase in the initiation-termination cycle of transcription. Cell 24 421-8 PubMed
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