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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 5. A natural 3′-OH RNA more stably maintains the post-translocation register of RNAP than 3′-H2 and 3′-OCH3 3′ ends, demonstrating the specificity of the exo III mapping assay for the RNAP TEC. GTPαS appears to stabilize the forward translocation state of the TEC slightly more strongly than GTP. GTP and GTPαS were added at 400 μM. The assay was at 40 mM KCl and pH 7.9.
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Figure 5: Figure 5. A natural 3′-OH RNA more stably maintains the post-translocation register of RNAP than 3′-H2 and 3′-OCH3 3′ ends, demonstrating the specificity of the exo III mapping assay for the RNAP TEC. GTPαS appears to stabilize the forward translocation state of the TEC slightly more strongly than GTP. GTP and GTPαS were added at 400 μM. The assay was at 40 mM KCl and pH 7.9.

Mentions: In Figure 1D, we show that, with a natural 3′- OH, the post-translocated A9 TEC can be observed at early times of incubation. The G8 TEC is mostly observed at the pre-translocated register within this time window (3–15 sec), and the G7 TEC appears to remain stubbornly post-translocated. Comparing the data in Figures 1C and 1D, for the chain terminated 3′dA9 and natural A9 TECs [lanes 7 and 8 (Fig. 1C) and 18 and 19 (Fig. 1D)], it appears that a RNA 3′-OH end may stabilize the post-translocated state slightly relative to a 3′-H2 chain-terminated end (also, see Figure 5 below). We conclude that the 3′dA9 TEC is highly suitable to observe strong NTP-dependent effects on RNAP TEC translocation, in order to preserve post-translocated TEC stability and to analyze transcriptional fidelity. Note that a 3′dA9 TEC + CTP just fills the 10 nt RNA+NTP/DNA hybrid channel (Fig. 1A).


Translocation and fidelity of Escherichia coli RNA polymerase.

Nedialkov YA, Burton ZF - Transcription (2013)

Figure 5. A natural 3′-OH RNA more stably maintains the post-translocation register of RNAP than 3′-H2 and 3′-OCH3 3′ ends, demonstrating the specificity of the exo III mapping assay for the RNAP TEC. GTPαS appears to stabilize the forward translocation state of the TEC slightly more strongly than GTP. GTP and GTPαS were added at 400 μM. The assay was at 40 mM KCl and pH 7.9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042587&req=5

Figure 5: Figure 5. A natural 3′-OH RNA more stably maintains the post-translocation register of RNAP than 3′-H2 and 3′-OCH3 3′ ends, demonstrating the specificity of the exo III mapping assay for the RNAP TEC. GTPαS appears to stabilize the forward translocation state of the TEC slightly more strongly than GTP. GTP and GTPαS were added at 400 μM. The assay was at 40 mM KCl and pH 7.9.
Mentions: In Figure 1D, we show that, with a natural 3′- OH, the post-translocated A9 TEC can be observed at early times of incubation. The G8 TEC is mostly observed at the pre-translocated register within this time window (3–15 sec), and the G7 TEC appears to remain stubbornly post-translocated. Comparing the data in Figures 1C and 1D, for the chain terminated 3′dA9 and natural A9 TECs [lanes 7 and 8 (Fig. 1C) and 18 and 19 (Fig. 1D)], it appears that a RNA 3′-OH end may stabilize the post-translocated state slightly relative to a 3′-H2 chain-terminated end (also, see Figure 5 below). We conclude that the 3′dA9 TEC is highly suitable to observe strong NTP-dependent effects on RNAP TEC translocation, in order to preserve post-translocated TEC stability and to analyze transcriptional fidelity. Note that a 3′dA9 TEC + CTP just fills the 10 nt RNA+NTP/DNA hybrid channel (Fig. 1A).

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