Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded - PubMed (original) (raw)

Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded

N Komissarova et al. Proc Natl Acad Sci U S A. 1997.

Abstract

RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked. Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of Escherichia coli RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3' end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme. The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage. Secondary structure elements or the 5' end of the transcript can prevent the isomerization by blocking the RNA threading. The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation.

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Figures

Figure 1

Arrest of ECs. After a 20-min incubation RNA-labeled EC12, EC27, and EC34 (lanes 1, 3, and 5) were allowed to resume elongation with the addition of the indicated NTPs (5 μM each) for 5 min and then were washed with TB (lanes 2, 4, and 6).

Figure 2

Effect of arrest on RNAP position on the DNA. (A) (Left) Dependence of EC27 inactivation on time and temperature. EC27 was allowed to resume elongation with the addition of 5 μM ATP, CTP, and UTP for 5 min after stalling under the indicated conditions. (Center and Right) ExoIII digestion of the nontemplate and template DNA strands (showing the front-end and rear-end footprints, respectively) in EC27 prepared after the indicated time of stalling. (B) (Left) ExoIII digestion of the nontemplate and template DNA strands in the active EC12, EC20, EC27, and EC34 and in the arrested EC27 and EC34. The arrested complexes were isolated in homogeneous form by adding the four NTPs prior to the digestion. (Right) Scheme summarizing the data of ExoIII footprinting. Vertical black lines symbolize the DNA strands, asterisks indicate the 5′ labeling of the strands, and ovals indicate RNAP. (C) (Left) Potassium permanganate (KMnO4) footprinting of the nontemplate DNA strand in the same complexes. Arrows indicate signals that originated from modified single-strand thymidines involved into the transcriptional bubble. (Right) Scheme summarizing the data of KMnO4 footprinting. The symbols are the same as above. Hexagons symbolize the transcriptional bubble. The positions of reactive and nonreactive T residues in the nontemplate strand are shown by the solid and open circles, respectively.

Figure 3

Effect of arrest on the transcript arrangement in RNAP. (A) RNase A footprinting of the transcript in active EC26 and in arrested EC27. The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. (B) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC20 and EC27 (lanes 6 and 4) and arrested fraction of EC27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

Figure 4

GreB-induced cleavage of the RNA in active and arrested complexes. (Right) Active EC26 and EC34 and homogeneous arrested EC27 and EC34 containing full-sized (lanes 1, 5, 9, and 13) or 5′-truncated transcripts (lanes 3, 7, 11, and 15) were labeled in the position +26A of the RNA (EC26, EC27, EC26T1, and EC27T1) or in position +34C (EC34 and 34T1; EC34T1 was obtained by walking with unlabeled EC26T1). The products of GreB cleavage were fractionated by centrifugation and the supernatants displayed the 3′-terminal fragments dissociated from the complexes. The arrows on the left side of the autoradiogram indicate the nontreated transcripts, and the brackets on the right side indicate the cleavage products. (Left) Scheme summarizing the results of the experiment. The large arrowheads show positions where GreB cleaves the RNA in the active and arrested complexes. The numbers indicate the size of corresponding segments of the transcripts derived from GreB-induced cleavage or from treatment with RNase T1. For other symbols, see Fig. 3.

Figure 5

Effect of oligonucleotides complementary to the 5′ part of the transcript on the efficiency of EC27 arrest and a model of transcriptional arrest. (A) Sequences of full-sized or truncated transcripts in EC27 and the set of overlapping complementary hexanucleotides (bold lines). The cylinders represent the zone of tight contacts between the enzyme and the product (see the text for detail). (B) EC27 was obtained from EC26 in the presence of the oligonucleotides and then chased to position +34. (C) A model of EC rearrangements during transcriptional arrest and the anti-arresting role of RNA secondary structure. The shaded triangle symbolizes the position of the active center (see the text for details).

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Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded - PubMed (original) (raw)