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RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement

Nature Structural & Molecular Biology volume 21, pages 794–802 (2014)Cite this article

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Abstract

The rates of RNA synthesis and the folding of nascent RNA into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA-exit channel can either increase pausing by interacting with RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain has been proposed to mediate some effects of nascent-RNA structures. However, the connections among RNA structure formation and RNAP clamp movement and catalytic activity remain uncertain. Here, we assayed exit-channel structure formation in Escherichia coli RNAP with disulfide cross-links that favor closed- or open-clamp conformations and found that clamp position directly influences RNA structure formation and RNAP catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening through interactions with the flap that slow translocation.

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Figure 1: Modest inhibition of RNA–RNA duplex formation by RNAP.
Figure 2: Effects of the flap-tip deletion on duplex-stabilized pausing and exit-channel duplex formation.
Figure 3: Effects of closed- and open-clamp cross-links on duplex formation and duplex-stabilized pausing.
Figure 4: Effects of pause-suppressing substitutions and RfaH on duplex formation and duplex-stabilized pausing.
Figure 5: asRNA, flap-tip, NusA and RfaH NTD effects on forward translocation.
Figure 6: Effects of clamp position, regulators and cap-region substitutions on exit-channel duplex formation and on pausing.

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Acknowledgements

We thank members of the Landick laboratory for many helpful discussions and for comments on the manuscript. This work was supported by US National Institutes of Health grants GM097458 to S.A.D. and GM038660 to R.L. We thank I. Artsimovitch (Ohio State University) for providing pIA777.

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Pyae P Hein, Kellie E Kolb, Tricia Windgassen, Michael J Bellecourt, Rachel A Mooney & Robert Landick

  2. Laboratory of Molecular Biophysics, Rockefeller University, New York, New York, USA

    Seth A Darst

  3. Department of Bacteriology, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Robert Landick

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Contributions

P.P.H., R.L., S.A.D. and R.A.M. designed the experiments. P.P.H., K.E.K., T.W., M.J.B. and R.A.M. performed experiments and data analysis. P.P.H. and R.L. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Formation of the 8-bp RNA–RNA duplex by pairing the 8-mer asRNA to the pyrrolo-cytosine (PC)-containing nascent RNA.

(a) Fluorescence excitation spectra of ssRNA (green), dsRNA (purple), ssRNA with 8mer non-complementary RNA (blue), ssRNA without PC (black), and buffer (orange) are shown. (b) Fluorescence emission spectra of ssRNA, dsRNA, ssRNA with 8mer non-complementary RNA, ssRNA without PC, and buffer are indicated and colored as in a. (c) Association and dissociation of the 8mer asRNA (dark purple; RNA-6598; Table S1) to PC-containing ssRNA (red; RNA-7604; Table S1) detected by changes in fluorescence intensity. A schematic of the assay is shown above the plot. Dissociation of the 8mer asRNA from the ssRNA is triggered by the presence of competitor RNA containing no PC (RNA-7418; Table S1). (d) Quenching of PC fluorescence is specific to duplex formation. Addition of a non-complementary RNA oligo (green. RNA-6753; Table S1) does not quench the fluorescence signal.

Supplementary Figure 2 Off-rate measurements of scaffold dissociation from the ePEC in the absence or presence of asRNA.

(a) Schematic of the experimental assay. E.coli RNAP (750 nM) was incubated with nucleic-acid scaffold (RNA #7604, tDNA #5420, and ntDNA #5069; Table S1) to form ePECs. Preformed ePECs (U19) were challenged with 100 μg heparin/mL in the presence or absence of asRNA. As a control, RNAP was preincubated with 100 μg heparin/mL followed by incubation with nucleic-acid scaffold (U19). Samples were separated on a native 4–15% gradient Phastgel (GE). (b) Native EMSA analysis of ePEC formation in the presence or absence of either asRNA or heparin. The formation of ePECs was detected by the slower electrophoretic mobility of the EC. Red star, 5´ 32P. (c) The efficiency of ePEC formation under various conditions was measured to determine the upper boundary for the ePEC dissociation rate (0.0004 s–1). (d) A model for asRNA binding to ePECs reflecting association of nascent RNA in the exit channel compared to the excluded alternate route of asRNA binding to transiently dissociated scaffold followed by reformation of ePEC. The maximum ePEC dissociation rate constant in either the absence or presence of asRNA was estimated from the EMSA analysis and is shown in red.

Supplementary Figure 3 Formation of cross-links with CSSC as an oxidant to stabilize open- and closed-clamp ECs, and the effects of cross-linking on duplex-stabilized pausing.

(a) Crosslink formation by the closed (top) and open (bottom) Cys pairs in ECs detected by non-reducing SDS-PAGE. Crosslinking was measured at different redox potentials generated with 0.8 mM DTT and varying concentrations of CSSC. (b) Crosslinked fraction of closed and open Cys-pair ECs plotted as a function of redox potential. The fractions crosslinked were determined as the fraction of retarded-mobility βS-Sβ´ relative to the total (β + β´ + βS-Sβ´). (c) ECs containing closed RNAP were formed, crosslinked, and extended to G21 in steps 1–6 (Online Methods). ECs were reconstituted on the nucleic-acid scaffold shown in Fig. 1a. For crosslinked samples, ECs were oxidized with 1 mM CSSC for 30 min at 37 °C (+CSSC; ~62– 66% crosslinked). G17 RNA was labeled and extended to U19 by addition of [α-32P] CTP and 100 μM UTP. The complexes were then incubated with or without 1 μM 8mer antisense RNA oligo for 10 min at 37 °C. ECs were elongated through the his pause site (U19) with 10 μM GTP. Samples were removed at 10, 20, 30, 40, 50, 60, 90, 120, 150, and 180 s and separated by denaturing PAGE. Remaining samples were incubated with 0.5 mM GTP and UTP each for an additional 3 min at the end of the reaction (“C” lane). (d) Representative pause assay gel panels for non-crosslinked and crosslinked open Cys-pair ECs. The lane marked “C” denotes chase lane in which ECs were incubated with 0.5 mM GTP and UTP for an additional 3 min after the time course. Open Cys-pair ECs oxidized with 2 mM CSSC and 0.8 mM DTT (Eh = – 0.22 V) exhibited over 60% crosslinking.

Supplementary Figure 4 Formation of cross-links with diamide as an oxidant to stabilize open- and closed-clamp ECs, and the effects of cross-linking on duplex-stabilized pausing.

(a) Crosslink formation in closed (top) and open (bottom) Cys-pair ECs detected by non-reducing SDS-PAGE. Crosslinking was measured at different concentrations of diamide generated with 0.8 mM DTT and varying concentrations of diamide. (b) Crosslinked fraction of closed and open Cys-pair ECs as a function of diamide concentrations. The fractions crosslinked were determined as the fraction of retarded-mobility βS-Sβ´ relative to the total (β + β´ + βS-Sβ´). (c) ECs containing closed Cys-pair RNAP were formed, crosslinked, and extended to G21 in steps 1-6 (Online Methods). ECs were reconstituted on nucleic-acid scaffold shown in Fig. 1a. For crosslinked samples, ECs were oxidized with 15 mM diamide for 30 min at 37 °C (+diamide; ~85–90% crosslinked). G17 RNA was labeled and extended to C18 by addition of [α-32P]CTP. The complexes were then incubated with or without 1 μM 8mer asRNA for 10 min at 37 °C. ECs were elongated through the his pause site (U19) with 10 μM GTP and 100 μM UTP. Samples were removed at 10, 20, 30, 40, 50, 60, 90, 120, 150, and 180 s and separated by denaturing PAGE. Remaining samples were incubated with 0.5 mM GTP and UTP each for an additional 3 min at the end of the reaction (“C” lane). (d) Representative pause assay gel panels for non-crosslinked and crosslinked open Cys-pair ECs. The lane marked “C” denotes chase lane in which ECs were incubated with 0.5 mM GTP and UTP for an additional 3 min after the time course. Open Cys-pair ECs oxidized with 15 mM diamide and 0.8 mM DTT exhibited ~86% crosslinking.

Supplementary Figure 5 Effects of closed- and open-clamp cross-linking on formation of exit-channel duplex.

(a) Model for association kinetics of asRNA binding to non-crosslinked ePEC and to the nucleic-acid scaffold shown in Fig. 1a. (b) Representative time traces show a decrease in fluorescence of PC upon addition of 5 μM (purple) and 15 μM (light green) asRNA to 250 nM non-crosslinked ePEC. Data were fit to model 1 shown in a. (c) Model for duplex formation in non-crosslinked Cys-pair ePECs, crosslinked closed Cys-pair ePECs, and on nucleic-acid scaffold. (d) Representative time traces show a decrease in fluorescence of PC upon addition of 5 μM (light blue) and 15 μM 8mer (black) asRNA to 250 nM closed crosslinked EC (~60% crosslinked). Data were fit the model 2 shown in b. (e) Effect of the closed-clamp crosslink on the rate of asRNA oligo binding to the nascent RNA. Fraction (f) of each species was determined from the quantification of the non-reducing SDS-PAGE and native EMSA samples as described in Online Methods. The concentration of EC in all experiments was 250 nM. The observed rate constant (kobs) of duplex formation for scaffold, non-crosslinked ECs, and crosslinked ECs was measured by PC fluorescence quenching upon addition of asRNA (see Online Methods). (f and g) Native EMSA analysis of EC formation. ECs were reconstituted on the nucleic-acid scaffold with wild-type and non-crosslinked or crosslinked closed or open Cys-pair E.coli RNAP. Samples were separated on a native 4-15% gradient Phastgel (GE). The positions of the free nucleic-acid scaffold (lane 1) and ECs are indicated. Reconstitution efficiency as measured by percentage shifted is indicated below each lane.

Supplementary Figure 6 UTP addition-rate measurements with rapid quench-flow analysis.

(a) EC scaffold (NT-8111, T-8103, RNA-8107; Table S1) used for reconstitution of ECs (yields C18 EC) and kinetic analysis of translocation following UMP incorporation, which is denoted by a lowercase letter. 6-MI was incorporated in the non-template DNA (NT) at the second nucleotide downstream from the active site of the U19 EC (+2 position), indicated by M and shown as white-on-purple. (b) Denaturing PAGE showing UMP incorporation by ECs incubated with or without asRNA. Samples were removed and quenched at 0.002, 0.004, 0.008, 0.016, 0.032, 0.064, 0.128, 0.256, and 1 s. (c) The 18mer RNA (C18) present in each lane in panel b was quantitated as a fraction of the total RNA in each lane and plotted as a function of reaction time. The rate of single-round UTP addition (kUTP) was obtained by fitting the disappearance of RNA19 to a single-exponential equation.

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Hein, P., Kolb, K., Windgassen, T. et al. RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement. Nat Struct Mol Biol 21, 794–802 (2014). https://doi.org/10.1038/nsmb.2867

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