Specific Recognition of the -10 Promoter Element by the Free RNA Polymerase σ Subunit

Anastasiya Sevostyanova,Andrey Feklistov,Nataliya Barinova,Ewa Heyduk,Irina Bass,Saulius Klimasauskas,Tomasz Heyduk,Andrey Kulbachinskiy

doi:10.1074/jbc.m702495200

Abstract

Bacterial RNA polymerase holoenzyme relies on its sigma subunit for promoter recognition and opening. In the holoenzyme, regions 2 and 4 of the sigma subunit are positioned at an optimal distance to allow specific recognition of the -10 and -35 promoter elements, respectively. In free sigma, the promoter binding regions are positioned closer to each other and are masked for interactions with the promoter, with sigma region 1 playing a role in the masking. To analyze the DNA-binding properties of the free sigma, we selected single-stranded DNA aptamers that are specific to primary sigma subunits from several bacterial species, including Escherichia coli and Thermus aquaticus. The aptamers share a consensus motif, TGTAGAAT, that is similar to the extended -10 promoter. We demonstrate that recognition of this motif by sigma region 2 occurs without major structural rearrangements of sigma observed upon the holoenzyme formation and is not inhibited by sigma regions 1 and 4. Thus, the complex process of the -10 element recognition by RNA polymerase holoenzyme can be reduced to a simple system consisting of an isolated sigma subunit and a short aptamer oligonucleotide.

Highlights

The ␴ subunit of bacterial RNA polymerase (RNAP)4 holoenzyme plays a key role in promoter recognition and opening
Structural Features of Aptamers Recognized by the ␴70 Subunit of E. coli RNA Polymerase—As the primary target for aptamer selection, we chose the ␴70 subunit from E. coli, which has a well defined promoter specificity and has been used as a
Control experiments demonstrated that free ␴70 did not bind nontemplate oligonucleotides containing the Ϫ10 promoter element (Kd Ͼ 10 ␮M)

Summary

EXPERIMENTAL PROCEDURES

Proteins—Wild-type and mutant E. coli ␴70, T. aquaticus, and Deinococcus radiodurans ␴A were obtained as described [26, 28, 29]. Selection of Aptamers to E. coli ␴70—The selection of aptamers was performed essentially as described previously [26]. The eluted DNA was amplified using primers corresponding to the fixed regions of the library (5Ј-GGGAGCTCAGAATAAACGCTCAA and BBB-5Ј-GATCCGGGCCTCATGTCGAA, where B is a biotin residue), DNA strands were separated by size on 10% denaturing PAGE, and the nonbiotinylated strand was purified and used for the round of selection. Oligonucleotides were 5Ј-end-labeled with [␥-32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences) and T4 polynucleotide kinase (New. was incubated with a series of dilutions of the ␴ subunit or its fragments (from 1 nM to 3 ␮M) in 50 ␮l of binding buffer for 30 min at room temperature; the samples were filtered through. The reaction was stopped after 5 min by adding an equal volume of stop buffer containing 2% SDS, 0.5 M Tris-HCl, pH 8.4, 100 mM.

Luminescence Resonance Energy Transfer Distance

RESULTS

Aptamer Kd TGTAGAAT TGCTAGAAT CCTAGAAT

DISCUSSION