Abstract
Prokaryotic RNA polymerase holoenzyme is composed of core subunits (alpha(2)betabeta'omega) plus a sigma factor that confers promoter specificity allowing for regulation of gene expression. Holoenzyme is known to undergo several conformational changes during the multiple steps of transcription initiation. However, the effects of these changes on the functions of specific regions have not been well characterized. In this work, we addressed the role of possible conformational change in region 2 of Escherichia coli sigma(70) by engineering disulfide bonds that "lock" region 2.1 with region 2.2 and region 2.2 with region 2.3. When these mutant holoenzymes were characterized for gross defects in multiple-round transcription, we found that insertion of either disulfide bond did not result in a fundamental block, indicating that the disulfide-containing holoenzymes are active. However, both disulfide-containing holoenzymes exhibited defects in formation and stability of the open complex. Our results suggest that conformational flexibility within sigma(70) region 2 facilitates open complex formation and transcription initiation.
Highlights
EXPERIMENTAL PROCEDURESAll of the reagents were purchased from Sigma unless otherwise indicated
RNA polymerase, a multi-subunit enzyme that is made up of five polypeptide chains (␣2Ј), is solely responsible for the synthesis of messenger, transfer, and ribosomal RNA in the bacterial cell
We addressed the role of possible conformational change in region 2 of Escherichia coli 70 by engineering disulfide bonds that “lock” region 2.1 with region 2.2 and region 2.2 with region 2.3
Summary
All of the reagents were purchased from Sigma unless otherwise indicated. Spectrophotometric grade glycerol was purchased from Fisher. E. coli LB culture medium contained 1.0% Bacto Tryptone, 0.5% Bacto Yeast Extract, and 0.5% NaCl. Ni-NTA buffer contained 25 mM Tris-HCl (pH 7.9), 50 mM NaCl, 5 mM imidazole, 0.1% Tween 20, and 5% glycerol. TE buffer contained 50 mM Tris-HCl (pH 7.9) and 0.5 mM EDTA. Storage buffer contained 50 mM Tris-HCl (pH 7.9), 100 mM NaCl, 0.1 mM EDTA, and 50% glycerol. A cysteine-less variant of 70 was made by the addition of C442S through site-directed mutagenesis using plasmid p[S442C]70 as template [28]. The region containing the substituted serines was subcloned by transferring the PstI-HindIII fragment into pHMK-His6-70 [29] to
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