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Bacterial RNA Polymerase Can Retain Σ Throughout Transcription

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Bacterial RNA Polymerase Can Retain Σ Throughout Transcription Bacterial RNA polymerase can retain σ70 throughout transcription Timothy T. Hardena,b, Christopher D. Wellsc, Larry J. Friedmanb, Robert Landickd,e, Ann Hochschildc, Jane Kondeva,1, and Jeff Gellesb,1 aDepartment of Physics, Brandeis University, Waltham, MA 02454; bDepartment of Biochemistry, Brandeis University, Waltham, MA 02454; cDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; dDepartment of Biochemistry, University of Wisconsin, Madison, WI 53706; and eDepartment of Bacteriology, University of Wisconsin, Madison, WI 53706 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved December 10, 2015 (received for review July 15, 2015) Production of a messenger RNA proceeds through sequential stages early elongation pause. This early elongation pause, in turn, al- of transcription initiation and transcript elongation and termination. lows loading of an antitermination factor that enables tran- During each of these stages, RNA polymerase (RNAP) function is scription of the late gene operon (10, 13–15). Similar promoter- regulated by RNAP-associated protein factors. In bacteria, RNAP- proximal pause elements are also associated with many E. coli associated σ factors are strictly required for promoter recognition promoters (16–19), but the function of these elements is yet and have historically been regarded as dedicated initiation factors. unknown. Furthermore, σ70 interaction sites on core RNAP par- However, the primary σ factor in Escherichia coli, σ70,canremain tially overlap with those of transcription elongation factors such as associated with RNAP during the transition from initiation to elon- NusA, NusG, and RfaH (20–23). This and other evidence raises the gation, influencing events that occur after initiation. Quantitative possibility that σ70 retained in TECs sterically occludes the binding studies on the extent of σ70 retention have been limited to com- of other factors, which in turn could affect processes modulated by plexes halted during early elongation. Here, we used multiwave- these factors, including intrinsic termination, rho-dependent ter- length single-molecule fluorescence-colocalization microscopy to mination, and transcription–translation coupling (10, 14, 15, 23). σ70– λ 70 observe the RNAP complex during initiation from the PR′ σ retention by TECs early in elongation (<100 bp downstream promoter and throughout the elongation of a long (>2,000-nt) of the promoter) is well established in vitro (9, 24). Retention transcript. Our results provide direct measurements of the fraction can be detected indirectly as pausing that occurs at downstream BIOCHEMISTRY 70 of actively transcribing complexes with bound σ and the kinetics pause elements that resemble promoter –10 elements (7, 12, 23). 70 70 of σ release from actively transcribing complexes. σ release In addition, TECs with σ70 stably bound have been reported (25), −1 from mature elongation complexes was slow (0.0038 s ); a substan- and retention of σ70 by TECs stalled at different positions down- 70 tial subpopulation of elongation complexes retained σ throughout stream of promoters has been confirmed in bulk (26, 27) and single- transcript elongation, and this fraction depended on the sequence molecule (28) studies. The latter data have been interpreted to of the initially transcribed region. We also show that elongation support models in which σ70 is stochastically released after pro- σ70 complexes containing manifest enhanced recognition of a pro- moter escape, but there are no studies directly characterizing moter-like pause element positioned hundreds of nucleotides release kinetics on actively elongating TECs. downstream of the promoter. Together, the results provide a quan- To understand the postinitiation roles of σ70, it is essential to σ70 titative framework for understanding the postinitiation roles of identify the conditions under which σ70 is retained by RNAP during transcription. after promoter escape and to describe the kinetics of its release from actively elongating TECs. Here, we use multiwavelength CoSMoS | single-molecule fluorescence | sigma factor | elongation complex | transcription regulation Significance lthough DNA-directed RNA synthesis can be carried out by In all kingdoms of life, gene transcription is not carried out by ARNA polymerase (RNAP) alone, it is well established that RNA polymerase enzymes alone. Instead, the behavior of RNA transcribing RNAPs in the cell have bound accessory proteins polymerases during transcription initiation, elongation, and that modulate transcription initiation and elongation (1). Eluci- termination is regulated by accessory proteins that bind to the dating the dynamics of accessory factor binding to and release polymerase molecule. Bacterial σ proteins are historically thought from the transcription apparatus is essential to achieving a quan- of as transcription initiation factors primarily involved in titative understanding of the molecular mechanisms that control promoter recognition. Here, we use light microscopy to di- transcription in cells. rectly observe the behavior of individual fluorescently labeled σ In bacteria, any of a variety of subunits can associate with the σ70 subunits during transcript elongation by Escherichia coli core RNAP, conferring on the enzyme the ability to bind to RNA polymerase. We show that σ70 can be retained on an RNA distinct subsets of promoter sequences (2). Some σ subunits re- polymerase molecule throughout transcription and alters poly- lease from core RNAP immediately upon the initiation of RNA merase behavior during transcript elongation. synthesis (3). In contrast, the primary σ factor in Escherichia coli, 70 σ , may be associated in vivo with a fraction of transcription Author contributions: T.T.H., C.D.W., A.H., J.K., and J.G. designed research; T.T.H. and C.D.W. elongation complexes (TECs) even far downstream of the pro- performed research; T.T.H., C.D.W., L.J.F., and R.L. contributed new reagents/analytic tools; – T.T.H. and L.J.F. analyzed data; T.T.H., J.K., and J.G. prepared the original draft and all moter (4 8). It is unclear whether this downstream association authors contributed to writing the final manuscript. in vivo reflects retention of the initiating σ70 subunit or binding The authors declare no conflict of interest. of σ70 after TEC formation and whether the σ70–TEC association This article is a PNAS Direct Submission. is kinetically stable during transcript elongation (9). σ70 Data deposition: The sequences reported in this paper have been deposited in the Gen- Retention of by early elongation TECs has demonstrated Bank database (accession nos. KT326913–KT326916). σ70 consequences for gene regulation. In particular, the -containing 1 70 To whom correspondence may be addressed. Email: [email protected] or gelles@ TEC (σ TEC) plays an essential role in bacteriophage λ late gene brandeis.edu. – σ70 expression (10 12) because bound is required for the rec- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ognition of a promoter-like pause element that induces a critical 1073/pnas.1513899113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1513899113 PNAS Early Edition | 1of6 Downloaded by guest on October 3, 2021 single-molecule fluorescence techniques to follow in real time probe spot, indicating the formation of a nascent transcript (Fig. the initiation and elongation of transcription complexes from the 1B). A control experiment without NTPs showed only 4% probe phage λ PR′ promoter. The measurements allow us to directly colocalization. The two intrinsic terminators near the down- observe σ70 retention on and subsequent departure from tran- stream end of the template (Fig. 1A) are expected to efficiently scription complexes, both near to and far (>2,000 nt) downstream induce rapid (within 1 s) release of the transcript from RNAP of the promoter, and to separately characterize the behavior of (31–33). Consistent with transcript release upon termination, TECs and σ70TECs with respect to elongation velocity, intrinsic 94% of transcript probe spots seen in the NTPs-containing sample termination efficiency, and –10-like pause element recognition. disappeared during the 47 min duration recording; the spots that disappeared (for example, Fig. 1C and Fig. S1,greentraces)hada Results median lifetime of 79 ± 34 s (±SE). The median Cy3–probe spot Direct Detection of σ70 on Actively Elongating Transcription Complexes. lifetime was not significantly altered by changes in laser exposure To observe the presence of σ70 within promoter complexes and TECs, (Fig. S2), indicating that most or all probe colocalizations were not we tethered linear DNA molecules labeled with AlexaFluor 488 prematurely terminated by photobleaching. During the median (AF488) dye and containing the phage λ PR′ promoter to the duration of the probe spot, an elongation complex is expected to surface of a glass flow chamber (Fig. 1A). We incubated the sur- transcribe 1260 ± 540 bp of DNA [at 15.9 ± 0.6 bp/s (34)]. Since face with a solution containing E. coli RNAP holoenzyme con- the template encodes a 2134- to 2322-nt long RNA, this analysis taining a σ70 subunit labeled on a single cysteine with Cy5 implies that the transcript is first detected by probe hybridization dye. Formation of promoter complexes was visualized as the ap- when the TEC is located 870 ± 540 bp downstream of the pro- pearance in total internal reflection fluorescence microscopy (29) moter, a value consistent
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