The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection

Abstract

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labeling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

Fig. 1

Using smFRET to investigate dynamics of RNAP open complexes. (a) Schematic of the hypothesis that dynamics of single-stranded DNA in the transcription bubble of the open complex allow RNAP to sample different transcriptional start sites. The positions of the RNAP active site (green oval) and the −10 element (orange downstream region) of the DNA are assumed to be fixed with respect to each other. Sampling of transcription start sites can therefore proceed via movement of single-stranded DNA within the transcription bubble, as indicated by the two representations of the open complex (RP_o). (b) Detecting RP_o formation with smFRET. dsDNA was labeled with donor and acceptor fluorophores on either side of the transcription bubble [donor at position −15 with respect to the +1 position and acceptor at position +15; see Supplementary Fig. 1 for the DNA sequence of lacCONS+2(A+2C) used in (b)]. smFRET spectroscopy combined with ALEX on diffusing molecules of dsDNA alone and dsDNA with RNAP (RP_o) was carried out. Ratio E* represents the uncorrected FRET efficiency, and curves were fitted with Gaussian functions to determine the center and width of the distributions.

Fig. 2

Start-site selection at lacCONS+2 and lacCONS+2 derivatives. (a) In vitro transcription reactions using lacCONS+2 promoters with base-pair substitutions; RNAP, dsDNA, ATP, UTP, CTP and [α³²P]GTP were incubated in transcription buffer at 37 °C for 5 min followed by heparin challenge and separation of the products on a polyacrylamide gel. Labeled RNA standards were used to determine the size of the products. (b) Sequences of the lacCONS+2 promoter and its derivatives. Boxes are drawn around the −10 and −35 elements and the +1 position is marked. The primary RNA products observed for each sequence are labeled in green.

Fig. 3

Transcription-bubble flexibility in RP_o. (a) Top panel: dsDNA, lacCONS+2(T-3A), labeled with donor and acceptor fluorophores at positions −15 and +15, respectively (with respect to the +1 position), was analyzed using smFRET. A FRET histogram was derived from the mean values of (at least) triplicate experiments. Sizable FRET distributions were fitted with a Gaussian function (black curve) to determine the center and width of the distribution. The calculated shot-noise-limited width is shown as a red Gaussian fit. Second panel: BVA of the FRET distribution of the dsDNA. Black arc represents static limit, colored contour plots represent frequency distributions (red contour, most abundant region; blue, less abundant) and triangles represent the standard deviation of a particular part of the two-dimensional histogram of the experimental data. Third panel: samples containing dsDNA and RP_o analyzed using smFRET. Fourth panel: samples containing dsDNA and RP_o analyzed using BVA. (b) Top and second panels: dsDNA labeled with donor and acceptor fluorophores at positions −5 and −3, respectively, was analyzed by smFRET and BVA. Third and fourth panels: samples containing dsDNA and RP_o analyzed by smFRET and BVA, respectively. (c) Top and second panels: dsDNA labeled with donor and acceptor fluorophores at positions −25 and −15, respectively, was analyzed by smFRET and BVA. Third and fourth panels: samples containing dsDNA and RP_o analyzed by smFRET and BVA, respectively.

Fig. 4

Start-site reprogramming changes the distance between DNA segments upstream and downstream of the transcription bubble. (a) Reprogramming by addition of initiating nucleotides. A lacCONS+2(G-2T;A+1C) promoter DNA fragment that can initiate transcription at positions −1 and +2 was mixed with RNAP to form open complexes (RP_o). The dsDNA promoter was labeled with fluorophores at positions −15 and +15 on either side of the transcription bubble. FRET histograms of RP_o alone (gray histogram), RP_o with GTP (blue histogram) and RP_o with ATP (green histogram) were overlaid (top panel). The difference between the FRET histograms of (RP_o +GTP) and (RP_o +ATP) was calculated (bottom panel). Both histograms were normalized to the area of the Gaussian fit function of the RP_o distribution. (b) Reprogramming by base-pair substitutions in the start-site region. A lacCONS+2(T-3A) promoter DNA fragment that initiates transcription at a range of positions from −2 to +3 was incubated with RNAP to form open complexes; these complexes were compared to open complexes formed using a lacCONS+2 promoter DNA fragment that initiates transcription from the +1 position. Both promoter fragments were labeled with fluorophores at positions −15 and +15 on either side of the transcription bubble. FRET histograms of RP_o of the lacCONS + 2 promoter (top panel) and lacCONS + 2(T-3A) promoter (lower panel) were compared.