Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 2. RNAP stalls in the post-translocation register. Forward translocation stability in the absence and presence of NTPs is strongly stimulated by increasing salt and decreasing pH. (A) Exo III mapping of 3′dA9 TECs +/− cognate CTP (400 μM) pH = 5.9, 6.9 and 7.9 and KCl = 40 and 150 mM. The scaffold is as in Figure 1B. (B) Exo III mapping of 3′dA9 TECs +/− cognate GTP or analogs (400 μM). Reactions are shown at KCl = 40, 60, 80, 100, 120 mM (scaffold as in Figure 1B but with a TDS-65 with 34C specifying GTP substrate and complementary 32G NDS-50 at RNA G10). GDP is < 2% contaminated with GTP, which is sufficient to extend A9 to G10 (lanes 8–11). RNA sequences for the two templates are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4042587&req=5

Figure 2: Figure 2. RNAP stalls in the post-translocation register. Forward translocation stability in the absence and presence of NTPs is strongly stimulated by increasing salt and decreasing pH. (A) Exo III mapping of 3′dA9 TECs +/− cognate CTP (400 μM) pH = 5.9, 6.9 and 7.9 and KCl = 40 and 150 mM. The scaffold is as in Figure 1B. (B) Exo III mapping of 3′dA9 TECs +/− cognate GTP or analogs (400 μM). Reactions are shown at KCl = 40, 60, 80, 100, 120 mM (scaffold as in Figure 1B but with a TDS-65 with 34C specifying GTP substrate and complementary 32G NDS-50 at RNA G10). GDP is < 2% contaminated with GTP, which is sufficient to extend A9 to G10 (lanes 8–11). RNA sequences for the two templates are shown.

Mentions: Histidine protonation is increased by lowering pH and raising salt.22 Elevating salt concentration is expected to support histidine protonation by stabilizing positive charge on the imidazole ring. Because a protonated H936 on the closed trigger loop might be expected to stabilize NTP binding by interaction with the β-phosphate9-11,23 (Fig. 1A; right panel), we considered the possibility that NTP-stabilized translocation might be stimulated by increasing salt and/or lowering pH. Here we show that the situation appears more complicated than a role for a protonated/deprotonated H936 in trigger loop dependent NTP loading and RNAP chemistry, because, somewhat surprisingly, both NTP-dependent and NTP-independent forward translocation appear to be strongly stabilized by raising salt into the physiological range and by lowering pH (Fig. 2A). The NTP-independent stabilization of the post-translocated TEC is not expected to depend on the protonation state of H936, which likely requires tight trigger loop closing on a cognate NTP. Observing the exo III footprint of the downstream RNAP boundary at a pH of 5.9, 6.9 and 7.9 and at a KCl concentration of 40 and 150 mM, the effects of lowering pH and/or of approaching physiological salt concentration appear strikingly similar. Both treatments strongly stabilize the forward translocation state of the RNAP TEC both in the absence and in the presence of 400 μM CTP. These results are consistent with a role for a protonated histidine residue (i.e., H936) in retention of CTP in the active site. Stabilization of the post-translocated state of the resting RNAP TEC, in the absence of CTP, however, cannot readily be attributed solely to H936, which might only be expected to interact with the NTP in a post-translocated TEC with a closed trigger loop.3,5 Conserved histidines that line the RNA/DNA channel and the downstream DNA/DNA duplex, therefore, may also be involved in regulating RNAP translocation (as discussed further below).


Translocation and fidelity of Escherichia coli RNA polymerase.

Nedialkov YA, Burton ZF - Transcription (2013)

Figure 2. RNAP stalls in the post-translocation register. Forward translocation stability in the absence and presence of NTPs is strongly stimulated by increasing salt and decreasing pH. (A) Exo III mapping of 3′dA9 TECs +/− cognate CTP (400 μM) pH = 5.9, 6.9 and 7.9 and KCl = 40 and 150 mM. The scaffold is as in Figure 1B. (B) Exo III mapping of 3′dA9 TECs +/− cognate GTP or analogs (400 μM). Reactions are shown at KCl = 40, 60, 80, 100, 120 mM (scaffold as in Figure 1B but with a TDS-65 with 34C specifying GTP substrate and complementary 32G NDS-50 at RNA G10). GDP is < 2% contaminated with GTP, which is sufficient to extend A9 to G10 (lanes 8–11). RNA sequences for the two templates are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Figure 2. RNAP stalls in the post-translocation register. Forward translocation stability in the absence and presence of NTPs is strongly stimulated by increasing salt and decreasing pH. (A) Exo III mapping of 3′dA9 TECs +/− cognate CTP (400 μM) pH = 5.9, 6.9 and 7.9 and KCl = 40 and 150 mM. The scaffold is as in Figure 1B. (B) Exo III mapping of 3′dA9 TECs +/− cognate GTP or analogs (400 μM). Reactions are shown at KCl = 40, 60, 80, 100, 120 mM (scaffold as in Figure 1B but with a TDS-65 with 34C specifying GTP substrate and complementary 32G NDS-50 at RNA G10). GDP is < 2% contaminated with GTP, which is sufficient to extend A9 to G10 (lanes 8–11). RNA sequences for the two templates are shown.
Mentions: Histidine protonation is increased by lowering pH and raising salt.22 Elevating salt concentration is expected to support histidine protonation by stabilizing positive charge on the imidazole ring. Because a protonated H936 on the closed trigger loop might be expected to stabilize NTP binding by interaction with the β-phosphate9-11,23 (Fig. 1A; right panel), we considered the possibility that NTP-stabilized translocation might be stimulated by increasing salt and/or lowering pH. Here we show that the situation appears more complicated than a role for a protonated/deprotonated H936 in trigger loop dependent NTP loading and RNAP chemistry, because, somewhat surprisingly, both NTP-dependent and NTP-independent forward translocation appear to be strongly stabilized by raising salt into the physiological range and by lowering pH (Fig. 2A). The NTP-independent stabilization of the post-translocated TEC is not expected to depend on the protonation state of H936, which likely requires tight trigger loop closing on a cognate NTP. Observing the exo III footprint of the downstream RNAP boundary at a pH of 5.9, 6.9 and 7.9 and at a KCl concentration of 40 and 150 mM, the effects of lowering pH and/or of approaching physiological salt concentration appear strikingly similar. Both treatments strongly stabilize the forward translocation state of the RNAP TEC both in the absence and in the presence of 400 μM CTP. These results are consistent with a role for a protonated histidine residue (i.e., H936) in retention of CTP in the active site. Stabilization of the post-translocated state of the resting RNAP TEC, in the absence of CTP, however, cannot readily be attributed solely to H936, which might only be expected to interact with the NTP in a post-translocated TEC with a closed trigger loop.3,5 Conserved histidines that line the RNA/DNA channel and the downstream DNA/DNA duplex, therefore, may also be involved in regulating RNAP translocation (as discussed further below).

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