Conformational Flexibility in ς70 Region 2 during Transcription Initiation

Abstract

Prokaryotic RNA polymerase holoenzyme is composed of core subunits (␣ 2 ␤␤) plus a 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 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 70 region 2 facilitates open complex formation and transcription initiation.RNA polymerase, a multi-subunit enzyme that is made up of five polypeptide chains (␣ 2 ␤␤Ј), is solely responsible for the synthesis of messenger, transfer, and ribosomal RNA in the bacterial cell. This enzyme has two distinct functional forms: core enzyme and holoenzyme. Although core enzyme is catalytically active and capable of transcription elongation, it is unable to initiate transcription at specific promoter sequences. Promoter recognition is accomplished by binding of the specificity factor , which positions holoenzyme on the promoter sequence (1, 2). Through the use of seven factors, Escherichia coli is able to direct transcription from multiple sets of promoter sequences, which confers the ability to regulate gene expression (3, 4). Amino acid sequence alignment of factors within the 70 family identified four regions of sequence homology (regions 1-4) (5). Region 2, which contains core binding and Ϫ10 promoter recognition determinants, accounts for the most sequence similarity among 70 family members. This suggests that this region plays an important role in transcription. Because 70 region 2 was shown to be a major interaction domain with the ␤Ј coiled-coil (6 -8), this region may be the site of functionally necessary conformational changes upon interaction with core enzyme.Based upon the sum of prior mechanistic, x-ray diffraction, electron microscopy, and luminescence resonance energy transfer studies, RNA polymerase core enzyme and 70 are thought to undergo multiple conformational changes through the process of transcription initiation: from binding, to formation of the closed complex, to stable open complex formation, to transcription initiation, and finally to factor release (3, 7, 9 -13). Evidence for intermediates during this process have been identified using the well studied lacUV5 promoter (12, 14 -16). The currently accepted pathway for open complex formation based on this promoter is shown below (15).R is RNA...

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