Report

Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching

See allHide authors and affiliations

Science  17 Nov 2006:
Vol. 314, Issue 5802, pp. 1139-1143
DOI: 10.1126/science.1131398

You are currently viewing the figures only.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

  1. Fig. 1.

    Background and experimental approach. (A) Models for RNAP–active-center translocation in abortive initiation. (Top) The “scrunching” model invokes a flexible element in DNA [(4, 7, 11, 12); see also (1319)]. In each cycle of abortive initiation, RNAP unwinds downstream DNA and pulls it into itself, accommodating the accumulated DNA as single-stranded bulges in the unwound region; upon release of the abortive RNA, RNAP extrudes the internalized DNA. (Middle) The “inchworming” model invokes a flexible element in RNAP (9, 10). In each cycle of abortive initiation, a module of RNAP containing the active center (white circle) detaches from the remainder of RNAP and translocates downstream; upon release of the abortive RNA, this module of RNAP reverse translocates. (Bottom) The “transient-excursions” model invokes abortive cycles that are transient–too short in lifetime and too infrequent in occurrence to be detected in a time-averaged, population-averaged approach, such as DNA footprinting (7). In each cycle of abortive initiation, RNAP translocates downstream as a unit; upon release of the abortive RNA, RNAP reverse translocates as a unit. (B) Experimental approach (fig. S1) (2022). The end-to-end extension of a mechanically stretched, negatively supercoiled (top) or positively supercoiled (bottom), single DNA molecule containing a single promoter is monitored. Unwinding of n turns of DNA by RNAP results in the compensatory loss of n negative supercoils or gain of n positive supercoils, and a readily detectable, nanometer-scale (n times 56 nm), movement of the bead.

  2. Fig. 2.

    Scrunching occurs in abortive initiation. (A) Single-molecule time traces and transition-amplitude histograms for RPo (0 NTPs) and RPitc,≤8 (ATP+UTP) at the N25 promoter. Data for positively and negatively supercoiled DNA are at the top and bottom, respectively. Green points, raw data (30 frames per s); red points, averaged data (1-s window); dashed lines in histograms, means; Δlobs,pos, transition amplitude with positively supercoiled DNA; Δlobs,neg, transition amplitude with negatively supercoiled DNA. (B) Transition-amplitude histogram for RPitc,≤1 (from control experiment providing only ATP). (C) Transition-amplitude histogram for RPitc,≤2 (from control experiment providing ATP, UTP, and rifampicin). (D) Differences in Δlobs,pos, Δlobs,neg, and unwinding relative to values in RPo (mean ± SE).

  3. Fig. 3.

    The extent of scrunching correlates with the length of the RNA product. (A) Transition-amplitude histograms for RPo (0 NTPs), RPitc,≤4 (ATP+UTP), and RPitc,≤8 (ATP+UTP+CTP) at the N25A5C promoter. Data for positively and negatively supercoiled DNA are at the top and bottom, respectively. (B) Differences in Δlobs,pos, Δlobs,neg, and unwinding relative to values in RPo (mean ± SE). (C) Prediction of scrunching model: number of base pairs scrunched equals N – 2, where N is the length of the RNA product. Sequence-specific RNAP-promoter interactions that define the upstream boundary of the unwound region are indicated by a gray box; RNAP structural elements that constrain the spacing between the upstream boundary of the unwound region and the RNAP active center are indicated by a gray bar; the RNAP active center is indicated by a white circle; the RNA product is in red; position +1 of the template DNA strand is in green; scrunched DNA nucleotides are in blue [(and are positioned as proposed in (25)].

  4. Fig. 4.

    Scrunching occurs in promoter escape in productive initiation. (A) Single-molecule time traces and transition-amplitude histograms for complete transcription cycles on N25-400-tR2 in the presence of all four NTPs. Data for positively and negatively supercoiled DNA are at the top and bottom, respectively. Transcription cycles are indicated by horizontal black bars; unwinding and rewinding transitions are indicated by numbered arrows (red numbered arrows for scrunching and reversal of scrunching); and states are indicated by horizontal lines and labeled on the right. (B) Differences in Δlobs,pos, Δlobs,neg, and unwinding relative to values in RPo. (C) Fraction of transcription cycles exhibiting at least one detectable scrunch (mean ± SEM; n = 100). (D) Distribution of scrunch lifetime [measured from midpoint of transition 2 to midpoint of transition 3 (n = 100)]. Error bars indicate statistical error.

Stay Connected to Science