Retention of Transcription Initiation Factor σ70 in Transcription Elongation: Single-Molecule Analysis

Department of Chemistry and Biochemistry, and California Nanosystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095, USA.
Molecular Cell (Impact Factor: 14.02). 12/2005; 20(3):347-56. DOI: 10.1016/j.molcel.2005.10.012
Source: PubMed


We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, approximately 70%-90%, of early elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region increases sigma70 retention in early elongation complexes. The results establish that a significant fraction, approximately 50%-60%, of mature elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region does not appreciably affect sigma70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma70, the half-life of sigma70 retention is long relative to the time-scale of elongation, suggesting that some complexes may retain sigma70 throughout elongation.

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Available from: Shimon Weiss, Oct 07, 2015
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    • "The observation that σ70 can induce pausing during transcription elongation suggested that it does not obligatory dissociate during the initiation-to-elongation transition. Indeed, FRET and chromatin-immunoprecipitation experiments demonstrated that a significant fraction of transcription elongation complexes (TECs) may contain the σ70 subunit both in vitro and in vivo, and supported a stochastic release model of σ70 dissociation from the TEC as a result of weakening σ-core interactions following initiation (17–21). Besides, in vitro experiments demonstrated that σ70 can rebind σ-free TEC and induce promoter-distal pausing when present at sufficiently high concentrations (11,22–24). "
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    ABSTRACT: A transcription initiation factor, the σ(70) subunit of Escherichia coli RNA polymerase (RNAP) induces transcription pausing through the binding to a promoter-like pause-inducing sequence in the DNA template during transcription elongation. Here, we investigated the mechanism of σ-dependent pausing using reconstituted transcription elongation complexes which allowed highly efficient and precisely controlled pause formation. We demonstrated that, following engagement of the σ subunit to the pause site, RNAP continues RNA synthesis leading to formation of stressed elongation complexes, in which the nascent RNA remains resistant to Gre-induced cleavage while the transcription bubble and RNAP footprint on the DNA template extend in downstream direction, likely accompanied by DNA scrunching. The stressed complexes can then either break σ-mediated contacts and continue elongation or isomerize to a backtracked conformation. Suppressing of the RNAP backtracking decreases pausing and increases productive elongation. On the contrary, core RNAP mutations that impair RNAP interactions with the downstream part of the DNA template stimulate pausing, presumably by destabilizing the stressed complexes. We propose that interplay between DNA scrunching and RNAP backtracking may have an essential role in transcription pausing and its regulation in various systems.
    Nucleic Acids Research 12/2011; 40(7):3078-91. DOI:10.1093/nar/gkr1158 · 9.11 Impact Factor
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    • "To explain how a promoter-proximal s 70 -dependent pause element increases the s 70 content of downstream elongation complexes, we propose that the sequencespecific interactions between s 70 and the pause element stabilize the association of s 70 with the early elongation complex during a ''critical window'' of nucleotide addition steps when the probability of s 70 release is relatively high. Support for the notion of such a critical window comes from the results of both ensemble and singlemolecule FRET measurements of the s 70 content of halted elongation complexes (Nickels et al. 2004; Kapanidis et al. 2005), which suggest that release of s 70 is biphasic, with an initial ''fast'' phase that occurs after synthesis of an RNA transcript ;12 nt in length and a subsequent ''slow'' phase. Thus, according to our model, the interaction between s 70 and an early elongation pause element stabilizes the association of s 70 with the RNAP core enzyme during critical nucleotide addition steps when s 70 release is ''fast'' (due, perhaps, to clashes between the nascent RNA and specific portions of s 70 ) (for review, see Mooney et al. 2005) and a significant fraction of the transcription complexes would otherwise release s 70 (Fig. 6B). "
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    ABSTRACT: The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (α(2)ββ'ω) in complex with a σ factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between σ and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of σ upon the transition to elongation. Furthermore, σ can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with σ also function to increase the σ content of downstream elongation complexes. Our findings establish σ-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.
    Genes & development 01/2011; 25(1):77-88. DOI:10.1101/gad.1991811 · 10.80 Impact Factor
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    • "), enabling high-throughput single-molecule spectroscopy of diffusing molecules. In particular, high-throughput two-color single-molecule detection would allow speeding up the acquisition of single-molecule FRET data, enabling the study of slow varying equilibria [41] or simply increasing the overall throughput of the powerful but still relatively tedious single-molecule fluorescence approaches [1,42]. "
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    ABSTRACT: We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed.
    Biomedical Optics Express 12/2010; 1(5):1408-1431. DOI:10.1364/BOE.1.001408 · 3.65 Impact Factor
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