Article

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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    • "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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    • "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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    • "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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