Identification of RNA Polymerase β′ Subunit Segment Contacting the Melted Region of the lacUV5 Promoter

Konstantin Brodolin,Arkady Mustaev,Konstantin Severinov,Vadim Nikiforov

doi:10.1074/jbc.275.5.3661

Konstantin Brodolin, Arkady Mustaev + Show 2 more

Open Access

https://doi.org/10.1074/jbc.275.5.3661

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Abstract

Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation. We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase beta' subunit contacting lacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of beta' and sigma(70) subunits at positions -5 and -3 on the nontemplate strand of the promoter DNA. These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex. The positions of formaldehyde cross-links in the beta' subunit were mapped to the N-terminal segment (Cys(198)-Met(237)), which is contiguous to the evolutionary conserved region B. The proximity of the beta' and sigma cross-links suggest that the N-terminal region of the beta' subunit, interacting with single-stranded promoter DNA, can cooperate with the sigma subunit in the process of open complex formation.

Highlights

Escherichia coli RNA polymerase (RNAP)1 is a multisubunit enzyme consisting of the catalytic core (␣2␤␤Ј) and ␴ subunit
In order to avoid these events in the current work, we reduced the time of crosslinking to 30 s at a 20 mM concentration of formaldehyde
The ␤Ј Subunit Segment Contacting Single-stranded DNA Near the Ϫ10 Consensus of the Promoter—Available data support the assumption that RNAP is involved in base-specific interactions with the single-stranded DNA of the promoter melted region [7, 18, 32, 33]

Summary

EXPERIMENTAL PROCEDURES

DNA and Proteins—E. coli RNAP containing a His tag in the ␤Ј subunit was isolated as described in Refs. 15 and 22. If the cross-linking was followed by fractionation on Ni2ϩ-NTA agarose (Qiagen), the reaction was stopped by addition of 2 volumes of 10 M urea. Bound cross-linking complexes were eluted in stop buffer and loaded on SDS-PAGE gels. For the cleavage at Cys residues the His-tagged ␤Ј-DNA crosslinked complexes were purified on Ni2ϩ-NTA agarose and treated with NTCBA [25] directly on the resin. The cross-linked complexes were separated by 5% SDS-PAGE, and gel slices containing radioactive bands were cut out and placed in Eppendorf tubes. Hydroxyl Radical Footprinting of Cross-linked Complexes—6 ␮g of RNAP was cross-linked with lacUV5 promoter in 40 ␮l of XLB, and SDS was added to 0.5% to terminate the reaction. The reaction was quenched by the addition of glycerol to 10% and stop buffer, and the solution was immediately loaded on SDS-PAGE gels. DNA was ethanol-precipitated, treated with 0.5 M piperidine, and analyzed on 6% sequencing gel

RESULTS

Position of Cys residue

DISCUSSION