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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 3. Exo III mapping to obtain a ranking of GTP analogs (400 μM) to stabilize forward RNAP TEC translocation at 40 (upper panel) and 150 mM KCl (lower panel). PPi was at 1 mM. pH is at 7.9.
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Figure 3: Figure 3. Exo III mapping to obtain a ranking of GTP analogs (400 μM) to stabilize forward RNAP TEC translocation at 40 (upper panel) and 150 mM KCl (lower panel). PPi was at 1 mM. pH is at 7.9.

Mentions: A potent effect of the incoming NTP on RNAP translocation has rarely been observed in exo III mapping experiments.2,17,18,20 By adjusting the length of the RNA, however, the response to cognate NTPs was enhanced, making exo III a more useful probe for the RNAP translocation state and for NTP loading (Figs. One and S1). Because NTP stabilization of forward translocation is robust (Fig. 1C), a spectrum of NTP analogs could be tested for their ability to stabilize forward translocation (see Figures 3 and 4 below). We previously showed that, for E. coli RNAP using exo III, a 10 nt RNA tends to be detected in a pre-translocated state, and, by comparison, a 11 nt RNA tends to backtrack more readily by one nt2. These results are consistent with X-ray crystal structures because, in Thermus thermophilus RNAP TECs, the RNA+NTP/DNA hybrid length is 10 nt4,5 (Fig. 1A). We reasoned, therefore, that a 10 nt RNA precisely fits the 10 nt channel hybrid length observed in structures, and therefore the TEC with a 10 nt RNA was observed primarily in the pre-translocated state. The TEC with a 11 nt RNA, by contrast, tended to backtrack by one nt to fill the 10 nt channel length, probably because a single nt was insufficient to stably occupy the RNA exit channel, allowing more rapid upstream sliding. We therefore considered the idea that exo III mapping experiments might be rendered more strongly NTP dependent if done on scaffolds with shorter RNA lengths that tend to resist backtracking and that may be more sensitive to cognate NTP addition.


Translocation and fidelity of Escherichia coli RNA polymerase.

Nedialkov YA, Burton ZF - Transcription (2013)

Figure 3. Exo III mapping to obtain a ranking of GTP analogs (400 μM) to stabilize forward RNAP TEC translocation at 40 (upper panel) and 150 mM KCl (lower panel). PPi was at 1 mM. pH is at 7.9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Figure 3. Exo III mapping to obtain a ranking of GTP analogs (400 μM) to stabilize forward RNAP TEC translocation at 40 (upper panel) and 150 mM KCl (lower panel). PPi was at 1 mM. pH is at 7.9.
Mentions: A potent effect of the incoming NTP on RNAP translocation has rarely been observed in exo III mapping experiments.2,17,18,20 By adjusting the length of the RNA, however, the response to cognate NTPs was enhanced, making exo III a more useful probe for the RNAP translocation state and for NTP loading (Figs. One and S1). Because NTP stabilization of forward translocation is robust (Fig. 1C), a spectrum of NTP analogs could be tested for their ability to stabilize forward translocation (see Figures 3 and 4 below). We previously showed that, for E. coli RNAP using exo III, a 10 nt RNA tends to be detected in a pre-translocated state, and, by comparison, a 11 nt RNA tends to backtrack more readily by one nt2. These results are consistent with X-ray crystal structures because, in Thermus thermophilus RNAP TECs, the RNA+NTP/DNA hybrid length is 10 nt4,5 (Fig. 1A). We reasoned, therefore, that a 10 nt RNA precisely fits the 10 nt channel hybrid length observed in structures, and therefore the TEC with a 10 nt RNA was observed primarily in the pre-translocated state. The TEC with a 11 nt RNA, by contrast, tended to backtrack by one nt to fill the 10 nt channel length, probably because a single nt was insufficient to stably occupy the RNA exit channel, allowing more rapid upstream sliding. We therefore considered the idea that exo III mapping experiments might be rendered more strongly NTP dependent if done on scaffolds with shorter RNA lengths that tend to resist backtracking and that may be more sensitive to cognate NTP addition.

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