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: 14.46). 12/2005; 20(3):347-56. DOI: 10.1016/j.molcel.2005.10.012
Source: PubMed

ABSTRACT 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, Jul 12, 2015
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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 · 12.64 Impact Factor
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    • "released from the RNAP core during elongation, it has sufficient affinity to remain bound during early transcription and thereby to induce pausing (Mooney et al., 2005, Kapanidis et al., 2005). In transcription from the λ late gene promoter λpR′, σ 70 region 2 binds a nontemplate −10-like element displaced 12 bp downstream from the promoter −10 element and induces a pause at +16 and +17, during which λQ protein acts (Grayhack et al., 1985). "
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    ABSTRACT: RNA polymerase of both bacteria and eukaryotes can stall or pause within tens of base pairs of its initiation site at the promoter, a state that may reflect important regulatory events in early transcription. In the bacterial model system, the σ(70) initiation factor stabilizes such pauses by binding a downstream repeat of a promoter segment, especially the '-10' promoter element. We first show here that the '-35' promoter element also can stabilize promoter-proximal pausing, through interaction with σ(70) region 4. We further show that an essential element of either type of pause is a sequence just upstream of the site of pausing that stabilizes RNA polymerase backtracking. Although the pause is not intrinsically backtracked, we suggest that the same sequence element is required both to stabilize the paused state and to potentiate backtracking.
    Molecular Microbiology 11/2010; 78(3):636-50. DOI:10.1111/j.1365-2958.2010.07347.x · 5.03 Impact Factor
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    • "The structural similarities between the aminoterminal domain of NusA and region 2 of s suggest that both proteins interact with the same region of RNAP to account for the mutual exclusivity of binding (Borukhov et al, 2005). However, elongation complexes can contain both s and NusA under certain circumstances (for example, Bar-Nahum & Nudler, 2001; Kapanidis et al, 2005; Mooney et al, 2009), suggesting that the mutually exclusive model is incorrect or that alternative conformations of s and NusA can be adopted for simultaneous interactions with RNAP. Here, we describe the structure of the B. subtilis NusA–RNAP complex that helps to explain how NusA regulates transcription by interacting with the emerging transcript around a region of RNAP known as the b-flap. "
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    ABSTRACT: There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are well-understood biochemically. However, despite the plethora of structural information made available on RNA polymerase in the last decade, little is available for RNA polymerase in complex with transcription elongation factors. To understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial RNA polymerase in complex with an essential transcription elongation factor. The resulting structure formed between the RNA polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation complex to an elongation complex, and how NusA is able to modulate transcription elongation and termination.
    EMBO Reports 08/2009; 10. DOI:10.1038/embor.2009.155 · 7.86 Impact Factor
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