A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing

6732–6740 Nucleic Acids Research, 2016, Vol. 44, No. 14 doi: 10.1093/nar/gkw285 Published online 20 April 2016 A universal transcription pause sequence is an element of initiation factor ␴70-dependent pausing Jeremy G. Bird, Eric J. Strobel and Jeffrey W. Roberts* Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA Received November 25, 2015; Revised April 5, 2016; Accepted April 7, 2016 ABSTRACT INTRODUCTION Transcription pausing at specific DNA regions is an important step in gene regulation in all organisms (1,2). A particularly well-understood example is the initiation factor ␴70-dependent pause that enables modification of Escherichia coli RNA polymerase (RNAP) by the bacteriophage lambda gene Q antiterminator protein (3,4). Within tens of nucleotides of transcription initiation, but well into the elongation phase, ␴70 in the RNAP transcription complex re-binds DNA at a near duplication of the original ‘−10’ promoter binding site, called the ‘−10-like sequence’ (5). This binding anchors a transcription pause that lasts on the order of tens of seconds, and provides a substrate for the Q protein to bind both RNAP and a Q binding element in the upstream DNA (6,7). ␴70-dependent promoterproximal pauses exist also in bacterial transcription (8–10), * To whom correspondence should be addressed. Tel: +1 607 255 2430; Fax: +1 607 255 6249; Email: Present addresses: Jeremy G. Bird, Departments of Genetics and Chemistry and Chemical Biology, and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA. Eric J. Strobel, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact The Escherichia coli ␴70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, ␴70 acts at other sites during transcription elongation as well. The pause is induced by ␴70 binding to a repeat of the promoter −10 sequence. After ␴70 binding, further RNA synthesis occurs as DNA is drawn (or ‘scrunched’) into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when ␴70 first engages the −10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the ‘elemental pause sequence’ recognized from its frequent occurrence in the E. coli genome. ␴70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause. and ␴70 can be shown to act during extended elongation in vivo and in vitro as well (11–13). The function of ␴70 in this pause is well characterized. Mutations in the −10-like sequence corresponding to mutations that impair function of the promoter −10 sequence also impair pausing (5). A mutationally altered ␴70 (L402F) selected to inhibit lambda late gene regulation impairs pausing during transcription in vitro and in vivo (14), although it is competent for initiation and does not prevent cell growth; a suppressor of L402F also enhances pausing (15). In addition, the paused complex has been characterized physically so that its structure is well understood (5,7,16,17). Although ␴70 is required for the pause, the actual site of pausing is not determined directly by the interaction of ␴70 and DNA. Binding of ␴70 regions 2 and 1.2 to DNA anchors the upstream edge of RNAP in a configuration like that of an open initiation complex; however, the catalytic center remains active, allowing polymerization of RNA to continue for 3–4 nucleotides after ␴70 has engaged (16). Analogous to the scrunching that has been demonstrated to occur during initial RNA synthesis from a promoter, downstream DNA at the pause is ‘scrunched’ into the enzyme as it is unwound (Figure 1A) (16,17). These findings raise the question of why scrunching in the paused complex stops where it does; what determines the actual RNA end in the paused complex? We show here that a G/C-rich segment at the upstream edge of the templating RNA/DNA hybrid in the transcribing enzyme largely determines the position of the pause in polymerization. The G/C-rich segment is part of a previously recognized universal pause-inducing sequence: the ‘elemental pause site’ (EPS), which traps the transcription complex from the pretranslocated position (18–20). Consistently, we find that other features characteristic of the EPS, especially the terminal RNA nucleotide, also strongly affect the strength of the ␴70-dependent pause. Nucleic Acids Research, 2016, Vol. 44, No. 14 6733 MATERIALS AND METHODS Plasmids and DNA templates Linear DNA templates for transcription were amplified from plasmids by polymerase chain reaction using primers indicated in supplemental materials and purified by gel extraction (Qiagen QIAquick gel extraction kit). All of the DNA templates were created by Agilent Quickchange mutagenesis from a p82pR’ +2G 1M −35-like mutant plasmid (equivalent to wild-type (WT) for these experiments) or the ␭pR’ pM650 plasmid (21,22). The 82pR’ +2G mutant was used throughout to prevent initiation stuttering from the WT initial AAA sequence; it is designated simply 82pR’ in the text. Heteroduplex templates were constructed as described by Ring et al. (5). Proteins In vitro transcription Reaction mixtures contained 2 nM DNA template, 10 nM RNAP holoenzyme (10 nM RNAP core reconstituted with 50 nM WT or mutant ␴70), 200 mM each of adenosine triphosphate, guanosine triphosphate and cytidine triphosphate, and 50 mM UTP (supplemented with 0.5 mCi/ml [a32 P]-UTP) in Transcription buffer (20 mM Tris–HCl (pH 8.0), 50 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin and 5% glycerol). Experiments shown in Figure 1B also contained 100 nM GreB in the reaction mixture. Reaction mixtures were first incubated at 37◦ C for 10 min to form open complexes. A single round of transcription was then initiated by the addition of MgCl2 to 5mM and rifampicin to 10 ␮g/ml and incubation continued at 37◦ C. Aliquots were taken at indicated times after the start of synthesis and mixed on ice with five volumes Stop solution (600 mM Tris–HCl (pH 8.0), 12 mM EDTA and 0.16 mg/ml tRNA). RNA was extracted by mixing with a equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). A total of 2.5 volumes of 100% ethanol was added to the aqueous layer to precipitate samples overnight at −20◦ C. Samples were run on (...truncated)