Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism

Abstract

Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a "scrunching" mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving "transient excursions" of RNAP relative to DNA or "inchworming" of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.

Fig. 1. Background and experimental approach

(A) Background. Three models have been proposed for RNAP-active-center translocation during initial transcription (-; see also 9-15): transient excursions, inchworming, and scrunching. White circles: RNAP active center; red dashed lines: RNA; black rectangles: promoter −10 and −35 elements. (B) Experimental approach. Top: Use of confocal microscopy with alternating-laser excitation (ALEX; 18-20) to monitor fluorescence of single transcription complexes. Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) diffuse through a femtoliter-scale observation volume (green; transit time ~1 ms); each molecule is illuminated with light that rapidly alternates between a wavelength that excites the donor and a wavelength that excites the acceptor. For each single molecule, and for each excitation wavelength, fluorescence emission is detected at both donor and acceptor emission wavelengths. This configuration permits calculation of two parameters: the donor-acceptor stoichiometry parameter, S, and the observed efficiency of donor-acceptor energy transfer, E* (18-20). The parameter S permits identification of molecules containing both donor and acceptor (S = 0.4-0.9; desired species; boxed in blue), molecules containing only a donor (S >0.9; undesired species, arising from the presence of free σ⁷⁰ molecules and buffer impurities), and molecules containing only an acceptor (S <0.4; undesired species, arising from the dissociation of non-specific complexes upon heparin challenge). Subsequent analysis is performed only on molecules containing both donor and acceptor. Bottom: Nucleoside-triphosphate (NTP) subsets and corresponding RNA products and complexes.

Fig. 2. Initial transcription does not involve transient excursions

(A) Experiment documenting movement of the RNAP leading edge relative to downstream DNA [tetramethylrhodamine as donor at σ⁷⁰ residue 366 (located in σR2, the σ⁷⁰ domain responsible for recognition of the promoter −10 element); Cy5 as acceptor at DNA position +20]. Top left, structural model of RP_o (29) showing positions of donor (green circle) and acceptor (red square). RNAP core is in gray; σ⁷⁰ is in yellow; the DNA template and nontemplate strands are in red and pink, respectively. Top right, E* histograms for RP_o and RP_itc,≤7. The vertical line and vertical dashed line mark mean E* values for RP_o and RP_itc,≤7, respectively. Bottom, predictions of the three models. (B) Experiment documenting absence of movement of the RNAP trailing edge relative to downstream DNA [tetramethylrhodamine as donor at σ⁷⁰ residue 569 (located in σR4, the σ⁷⁰ domain responsible for recognition of the promoter −35 element); Cy5 as acceptor at DNA position −39]. Subpanels as in (A).

Fig. 3. Initial transcription does not involve inchworming

(A) Experiment documenting absence of movement of the RNAP leading edge relative to −10/−35 spacer DNA [tetramethylrhodamine as donor at σ⁷⁰ residue 366 (located in σR2, the σ⁷⁰ domain responsible for recognition of the promoter −10 element); Alexa647 as acceptor at DNA position −20]. Subpanels as in Fig. 2A. (B) Experiment documenting absence of movement of the RNAP trailing edge relative to −10/−35 spacer DNA [tetramethylrhodamine as donor at σ⁷⁰ residue 569 (located in σR4, the σ⁷⁰ domain responsible for recognition of the promoter −35 element); Alexa647 as acceptor at DNA position −20]. Subpanels as in Fig. 2A.

Fig. 4. Initial transcription involves scrunching

(A) Experiment documenting contraction of DNA between positions −15 and +15 [Cy3B as donor at DNA position −15; Alexa647 as acceptor at DNA position +15]. Subpanels as in Fig. 2A. [The two donor-acceptor species in the E* histograms comprise free DNA (lower-E* species) and RP_o or RP_itc,≤7 (higher-E* species; higher FRET attributable to RNAP-induced DNA bending). Free DNA is present in all experiments, arising from dissociation of nonspecific complexes upon heparin challenge during preparation of RP_o, but is detected only in this experiment because DNA contains both donor and acceptor only in this experiment.] (B) Summary of results. Structural model of RP_o (29) showing all donor-acceptor distances monitored in this work (Figs 2-4A; S2-S8). Distances that remain unchanged upon transition from RP_o to RP_itc,≤7, are indicated with thin blue lines. Distances that decrease upon transition from RP_o to RP_itc,≤7, are indicated with thick blue lines. The red and pink arrows show the proposed positions at which scrunched template-strand DNA and scrunched nontemplate-strand DNA, respectively, emerge from RNAP (i.e., near template-strand positions −9 to −10 and near nontemplate-strand positions −5 to −6).