Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and RNA polymerase holoenzyme associate to form a 2:2 complex in vitro. No complexes of lower stoichiometry (1:1, 2:1, 1:2) were detected over a wide range of CAP and RNA polymerase concentrations, suggesting that the interaction is highly cooperative. The absence of higher stoichiometry complexes, even in the limit of high [protein], suggests that the 2:2 species represents binding saturation for this system. The 2:2 pattern of complex formation is robust. A lower-limit estimate of the formation constant in our standard buffer (40 mM Tris (pH 7.9), 10 mM MgCl 2 , 0.1 mM dithiothreitol, 5% glycerol, 100 mM KCl) is 2 ؋ 10 20 M ؊3 . The qualitative pattern of association is unchanged over the temperature range 4°C < T < 20°C, by substitution of glutamate for chloride as the dominant anion, or on addition of 20 M cAMP to the reaction mix. These results limit the possible mechanisms of CAP-polymerase association. In addition, they support the idea that CAP binding may influence the availability of the monomeric form of RNA polymerase that mediates transcription at many promoters.

Section: Resultssupporting

confidence: 80%

supporting

confidence: 92%

mentioning

confidence: 99%

See 1 more Smart Citation

The Escherichia coli Cyclic AMP Receptor Protein Forms a 2:2 Complex with RNA Polymerase Holoenzyme, in Vitro

Dyckman

Fried

2002

“…A small, variable fraction of the transcription complexes containing biotinated RNAP were observed to self-associate under the low ionic strength electrophoresis conditions, yielding a slower mobility band that was independent of the presence of streptavidin. Self-association of wildtype RNAP at low salt has been previously reported (41,42).…”

mentioning

confidence: 82%

Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

Nørrelykke

Engh

Landick

et al. 2004

102

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogenous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a ϳ2650-bp DNA during transcript elongation in vitro at 0.5 mM nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, ‫؍‬ 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.Different individual molecules within a purified enzyme or ribozyme preparation can display greatly differing rate constants for catalytic turnover (1-3). Some of this variability may be due to the presence of enzyme molecules with differing covalent structures; these may arise from the presence of two or more related genes, from alternative splicing of a single pre-mRNA, or from post-translational protein modifications (4). However, some heterogeneity may also arise from the adoption by enzyme molecules with identical covalent structures of different conformational states that persist through multiple catalytic turnovers (5, 6). It has been proposed that such conformational heterogeneity may be a heretofore unappreciated feature of macromolecular catalysts in general (2).Bacterial RNA polymerases (RNAPs), 1 and their structurally and mechanistically similar homologs, eukaryotic RNAP II enzymes, are large, multisubunit proteins that synthesize all cellular mRNAs (7,8). These enzymes are processive; a given RNA molecule is synthesized in its entirety by the same RNAP molecule. In bacterial RNAPs, most RNA synthesis takes place in a stable transcription elongation complex (TEC) containing the core RNAP subunits, the template DNA, and the nascent RNA transcript. The TEC structure has been extensively characterized (9 -12). A variety of proteins and DNA sequence elements in both prokaryotes and eukaryotes regulate gene expression by altering the extent to which active TECs terminate transcription upstream of or within genes (13-15). The efficiency of termi...

“…Both circular dichroism and NMR require high protein concentrations (micromolar to millimolar) at which RNA polymerase has been shown to aggregate (17,18), making these spectroscopic approaches not useful for analyzing this system. Methods previously utilized to address conformational changes in RNA polymerase holoenzyme, such as fluorescence resonance energy transfer (FRET) and thermodynamic measurements (9,13), allowed the observation of gross conformational changes but were not able to examine specific regions of the proteins or changes during the sequential steps of transcription.…”

mentioning

confidence: 99%

Conformational Flexibility in ς70 Region 2 during Transcription Initiation

Anthony

Burgess²

2002

Self Cite

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...