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Translocation and fidelity of Escherichia coli RNA polymerase.

Nedialkov YA, Burton ZF - Transcription (2013)

Bottom Line: The forward translocation state is made more stable by lowering the pH and/or by elevating the salt concentration, indicating a probable role of protonated histidine(s) in regulating accurate NTP loading and translocation.Because the post-translocated TEC can be strongly stabilized by NTP addition, NTP analogs were ranked for their ability to preserve the post-translocation state, giving insight into RNAP fidelity.Effects of NTPs (and analogs) and analysis of chemically modified RNA 3' ends demonstrate that patterns of exo III mapping arise from intrinsic and subtle alterations at the RNAP active site, far from the site of exo III action.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology; Michigan State University; E. Lansing, MI USA.

ABSTRACT
Exonuclease (exo) III was used as a probe of the Escherichia coli RNA polymerase (RNAP) ternary elongation complex (TEC) downstream border. In the absence of NTPs, RNAP appears to stall primarily in a post-translocated state and to return slowly to a pre-translocated state. Exo III mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation, in which RNAP freely and rapidly oscillates between pre- and post-translocated positions. The forward translocation state is made more stable by lowering the pH and/or by elevating the salt concentration, indicating a probable role of protonated histidine(s) in regulating accurate NTP loading and translocation. Because the post-translocated TEC can be strongly stabilized by NTP addition, NTP analogs were ranked for their ability to preserve the post-translocation state, giving insight into RNAP fidelity. Effects of NTPs (and analogs) and analysis of chemically modified RNA 3' ends demonstrate that patterns of exo III mapping arise from intrinsic and subtle alterations at the RNAP active site, far from the site of exo III action.

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Figure 1. A 9 nt RNA + NTP length gives very strong NTP stabilization of the post-translocation state of the RNAP TEC. (A) Nucleotide scaffolds for pre- and post-translocated TECs (PRE and PST). Template DNA strand (TDS) is blue; non-template DNA strand (NDS) is green; RNA is red. The NTP substrate (red) is in stick representation. Mg2+ is magenta. The closed trigger loop (TL) is yellow. β’ H936 is cyan. The image was derived from PDB 205J5 and drawn using Visual Molecular Dynamics.38 (B) Schematic of experiments for downstream border exo III mapping at TEC-G8 and TEC-A9. * indicates a 32P radiolabel; # indicates a sulfur for oxygen substitution in the TDS to block exo III (orange) digestion. Arrows indicate the upstream to downstream direction of transcription. The positions of the i and i+1 sites are indicated for pre- and post-translocated TECs. At 40 mM KCl, exo III digestion is blocked primarily at the i+18 position. At higher KCl and/or lower pH, digestion can be slowed at i+19 and i+18 (see below). As in panel A, the TDS is blue, the NDS is green and the RNA is red. The TEC bubble is indicated in outlined letters and pink shading. (C) Effects of NTPs (100 μM ATP or CTP) on chain-terminated 3′dG8 and 3′dA9 TECs. Exo III reaction times are in seconds (s). (D) Translocation of G7, G8 and A9 TECs (no chain termination). KCl is 40 mM; pH is 7.9. Backtracked (BTR), pre- (PRE) and post-translocated (PST) TECs are indicated.
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Figure 1: Figure 1. A 9 nt RNA + NTP length gives very strong NTP stabilization of the post-translocation state of the RNAP TEC. (A) Nucleotide scaffolds for pre- and post-translocated TECs (PRE and PST). Template DNA strand (TDS) is blue; non-template DNA strand (NDS) is green; RNA is red. The NTP substrate (red) is in stick representation. Mg2+ is magenta. The closed trigger loop (TL) is yellow. β’ H936 is cyan. The image was derived from PDB 205J5 and drawn using Visual Molecular Dynamics.38 (B) Schematic of experiments for downstream border exo III mapping at TEC-G8 and TEC-A9. * indicates a 32P radiolabel; # indicates a sulfur for oxygen substitution in the TDS to block exo III (orange) digestion. Arrows indicate the upstream to downstream direction of transcription. The positions of the i and i+1 sites are indicated for pre- and post-translocated TECs. At 40 mM KCl, exo III digestion is blocked primarily at the i+18 position. At higher KCl and/or lower pH, digestion can be slowed at i+19 and i+18 (see below). As in panel A, the TDS is blue, the NDS is green and the RNA is red. The TEC bubble is indicated in outlined letters and pink shading. (C) Effects of NTPs (100 μM ATP or CTP) on chain-terminated 3′dG8 and 3′dA9 TECs. Exo III reaction times are in seconds (s). (D) Translocation of G7, G8 and A9 TECs (no chain termination). KCl is 40 mM; pH is 7.9. Backtracked (BTR), pre- (PRE) and post-translocated (PST) TECs are indicated.

Mentions: The nucleic acid scaffolds for RNA polymerase (RNAP) pre- and post-translocated ternary elongation complexes (TECs) are depicted in Figure 1A. RNAP appears to rest in a primarily post-translocated state and to return to the pre-translocated state slowly and reversibly.1,2 This conclusion has been advanced based on X-ray crystal structures of RNAP TECs, which are mostly post-translocated.3-8 Fluorescence changes associated with translocation also indicate this conclusion,1 as do exonuclease (exo) III mapping studies of TEC boundaries.2 Here, we support this model by tuning the exo III mapping experiment and stabilizing the post-translocation state, using varied RNA length, lowered pH and salt in the physiological range. Multi-subunit RNAPs do not appear to rapidly oscillate between pre- and post-translocation positions. The transition from the pre- to the post-translocated state must be rapid, to account for rapid elongation rates; but as we show here, the return to the pre-translocated state is much slower and tunable by strengthening or weakening RNAP-nucleic acid interactions.


Translocation and fidelity of Escherichia coli RNA polymerase.

Nedialkov YA, Burton ZF - Transcription (2013)

Figure 1. A 9 nt RNA + NTP length gives very strong NTP stabilization of the post-translocation state of the RNAP TEC. (A) Nucleotide scaffolds for pre- and post-translocated TECs (PRE and PST). Template DNA strand (TDS) is blue; non-template DNA strand (NDS) is green; RNA is red. The NTP substrate (red) is in stick representation. Mg2+ is magenta. The closed trigger loop (TL) is yellow. β’ H936 is cyan. The image was derived from PDB 205J5 and drawn using Visual Molecular Dynamics.38 (B) Schematic of experiments for downstream border exo III mapping at TEC-G8 and TEC-A9. * indicates a 32P radiolabel; # indicates a sulfur for oxygen substitution in the TDS to block exo III (orange) digestion. Arrows indicate the upstream to downstream direction of transcription. The positions of the i and i+1 sites are indicated for pre- and post-translocated TECs. At 40 mM KCl, exo III digestion is blocked primarily at the i+18 position. At higher KCl and/or lower pH, digestion can be slowed at i+19 and i+18 (see below). As in panel A, the TDS is blue, the NDS is green and the RNA is red. The TEC bubble is indicated in outlined letters and pink shading. (C) Effects of NTPs (100 μM ATP or CTP) on chain-terminated 3′dG8 and 3′dA9 TECs. Exo III reaction times are in seconds (s). (D) Translocation of G7, G8 and A9 TECs (no chain termination). KCl is 40 mM; pH is 7.9. Backtracked (BTR), pre- (PRE) and post-translocated (PST) TECs are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Figure 1. A 9 nt RNA + NTP length gives very strong NTP stabilization of the post-translocation state of the RNAP TEC. (A) Nucleotide scaffolds for pre- and post-translocated TECs (PRE and PST). Template DNA strand (TDS) is blue; non-template DNA strand (NDS) is green; RNA is red. The NTP substrate (red) is in stick representation. Mg2+ is magenta. The closed trigger loop (TL) is yellow. β’ H936 is cyan. The image was derived from PDB 205J5 and drawn using Visual Molecular Dynamics.38 (B) Schematic of experiments for downstream border exo III mapping at TEC-G8 and TEC-A9. * indicates a 32P radiolabel; # indicates a sulfur for oxygen substitution in the TDS to block exo III (orange) digestion. Arrows indicate the upstream to downstream direction of transcription. The positions of the i and i+1 sites are indicated for pre- and post-translocated TECs. At 40 mM KCl, exo III digestion is blocked primarily at the i+18 position. At higher KCl and/or lower pH, digestion can be slowed at i+19 and i+18 (see below). As in panel A, the TDS is blue, the NDS is green and the RNA is red. The TEC bubble is indicated in outlined letters and pink shading. (C) Effects of NTPs (100 μM ATP or CTP) on chain-terminated 3′dG8 and 3′dA9 TECs. Exo III reaction times are in seconds (s). (D) Translocation of G7, G8 and A9 TECs (no chain termination). KCl is 40 mM; pH is 7.9. Backtracked (BTR), pre- (PRE) and post-translocated (PST) TECs are indicated.
Mentions: The nucleic acid scaffolds for RNA polymerase (RNAP) pre- and post-translocated ternary elongation complexes (TECs) are depicted in Figure 1A. RNAP appears to rest in a primarily post-translocated state and to return to the pre-translocated state slowly and reversibly.1,2 This conclusion has been advanced based on X-ray crystal structures of RNAP TECs, which are mostly post-translocated.3-8 Fluorescence changes associated with translocation also indicate this conclusion,1 as do exonuclease (exo) III mapping studies of TEC boundaries.2 Here, we support this model by tuning the exo III mapping experiment and stabilizing the post-translocation state, using varied RNA length, lowered pH and salt in the physiological range. Multi-subunit RNAPs do not appear to rapidly oscillate between pre- and post-translocation positions. The transition from the pre- to the post-translocated state must be rapid, to account for rapid elongation rates; but as we show here, the return to the pre-translocated state is much slower and tunable by strengthening or weakening RNAP-nucleic acid interactions.

Bottom Line: The forward translocation state is made more stable by lowering the pH and/or by elevating the salt concentration, indicating a probable role of protonated histidine(s) in regulating accurate NTP loading and translocation.Because the post-translocated TEC can be strongly stabilized by NTP addition, NTP analogs were ranked for their ability to preserve the post-translocation state, giving insight into RNAP fidelity.Effects of NTPs (and analogs) and analysis of chemically modified RNA 3' ends demonstrate that patterns of exo III mapping arise from intrinsic and subtle alterations at the RNAP active site, far from the site of exo III action.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology; Michigan State University; E. Lansing, MI USA.

ABSTRACT
Exonuclease (exo) III was used as a probe of the Escherichia coli RNA polymerase (RNAP) ternary elongation complex (TEC) downstream border. In the absence of NTPs, RNAP appears to stall primarily in a post-translocated state and to return slowly to a pre-translocated state. Exo III mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation, in which RNAP freely and rapidly oscillates between pre- and post-translocated positions. The forward translocation state is made more stable by lowering the pH and/or by elevating the salt concentration, indicating a probable role of protonated histidine(s) in regulating accurate NTP loading and translocation. Because the post-translocated TEC can be strongly stabilized by NTP addition, NTP analogs were ranked for their ability to preserve the post-translocation state, giving insight into RNAP fidelity. Effects of NTPs (and analogs) and analysis of chemically modified RNA 3' ends demonstrate that patterns of exo III mapping arise from intrinsic and subtle alterations at the RNAP active site, far from the site of exo III action.

Show MeSH
Related in: MedlinePlus