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Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre.

Roghanian M, Yuzenkova Y, Zenkin N - Nucleic Acids Res. (2011)

Bottom Line: Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript.Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre.This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne NE2 4AX, UK.

ABSTRACT
The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

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(A) Upon folding the TL forms part of the RNAP active centre. The TL of T. thermophilus RNAP [PDB 2BE5 and 2O5J, respectively (7,34)], which has complete conservation with the active centre amino acids of T. aquaticus RNAP used in our study, in unfolded and folded states is coloured green and cyan, respectively. The amino acids of the TL that form the ‘TL active centre’ upon TL folding (M1238, R1239, H1242, T1243) are shown in space fill in both folded and unfolded states of the TL. The amino acids (not belonging to the TL) that surround catalytic Mg2+ ions and the incoming NTP are shown in grey spacefill. The aspartate triad is shown as purple sticks. Mg2+ ions of the active centre are shown as spheres. Incoming NTP, RNA 3′-end and DNA template bases are shown as orange, red and black sticks, respectively. The N-terminal coiled-coil domain of Gre factor (structure of E. coli GreA was used; PDB 1GRJ) which, according to our results, substitutes for the TL upon misincorporation or backtracking (see main text), was positioned to reach the active centre of RNAP without significant clashes with surrounding structure, and is shown as yellow ribbons. (B) Elongation complexes used in this study. Shown are the sequences of RNA, template strand (T DNA) and non-template strand (NT DNA) used to assemble elongation complexes (see ‘Materials and Methods’ section). (C) Gre-catalysed hydrolysis is independent of the TL. Kinetics of the cleavage reaction (also schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (red) and ΔTL RNAP (blue), in the absence (left) or presence (right) of Gre factor. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). The grey arrows represent the contributions of the TL and Gre to the rate of the reaction. Representative gels for cleavage by WT and ΔTL RNAPs in the absence or the presence of Gre are shown below the plots. Note that the kinetics of Gre catalysed reactions were measured at 20°C to reduce the rate of the reaction to allow it to be measured manually. (D) Gre-catalysed hydrolysis is independent of the H1242 of the TL. Kinetics of the cleavage reaction in mEC15 (as in panel C) by H1242A RNAP with and without Gre, and WT RNAP with Gre. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (E) The TL is not involved in Mg2+ chelation during phosphodiester bond hydrolysis. Dependence of kobs on the Mg2+ concentration during intrinsic cleavage (as in panel C) by WT (red) and ΔTL (blue) RNAPs. The values of apparent Km[Mg2+] are shown below the plots. The values of kobs at different Mg2+ concentrations were calculated by single exponential fitting of the kinetics data (as in panel C) and Km[Mg2+] values were calculated as described in ‘Materials and Methods’ section. (F) pH profiles of the reactions described in panel C in the absence and the presence of Gre. The values of kobs at different pH were calculated by single exponential fitting of the kinetics data (see panel C and ‘Materials and Methods’ section). Note that reactions with Gre were performed at 20°C (as in panel C).
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Figure 1: (A) Upon folding the TL forms part of the RNAP active centre. The TL of T. thermophilus RNAP [PDB 2BE5 and 2O5J, respectively (7,34)], which has complete conservation with the active centre amino acids of T. aquaticus RNAP used in our study, in unfolded and folded states is coloured green and cyan, respectively. The amino acids of the TL that form the ‘TL active centre’ upon TL folding (M1238, R1239, H1242, T1243) are shown in space fill in both folded and unfolded states of the TL. The amino acids (not belonging to the TL) that surround catalytic Mg2+ ions and the incoming NTP are shown in grey spacefill. The aspartate triad is shown as purple sticks. Mg2+ ions of the active centre are shown as spheres. Incoming NTP, RNA 3′-end and DNA template bases are shown as orange, red and black sticks, respectively. The N-terminal coiled-coil domain of Gre factor (structure of E. coli GreA was used; PDB 1GRJ) which, according to our results, substitutes for the TL upon misincorporation or backtracking (see main text), was positioned to reach the active centre of RNAP without significant clashes with surrounding structure, and is shown as yellow ribbons. (B) Elongation complexes used in this study. Shown are the sequences of RNA, template strand (T DNA) and non-template strand (NT DNA) used to assemble elongation complexes (see ‘Materials and Methods’ section). (C) Gre-catalysed hydrolysis is independent of the TL. Kinetics of the cleavage reaction (also schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (red) and ΔTL RNAP (blue), in the absence (left) or presence (right) of Gre factor. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). The grey arrows represent the contributions of the TL and Gre to the rate of the reaction. Representative gels for cleavage by WT and ΔTL RNAPs in the absence or the presence of Gre are shown below the plots. Note that the kinetics of Gre catalysed reactions were measured at 20°C to reduce the rate of the reaction to allow it to be measured manually. (D) Gre-catalysed hydrolysis is independent of the H1242 of the TL. Kinetics of the cleavage reaction in mEC15 (as in panel C) by H1242A RNAP with and without Gre, and WT RNAP with Gre. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (E) The TL is not involved in Mg2+ chelation during phosphodiester bond hydrolysis. Dependence of kobs on the Mg2+ concentration during intrinsic cleavage (as in panel C) by WT (red) and ΔTL (blue) RNAPs. The values of apparent Km[Mg2+] are shown below the plots. The values of kobs at different Mg2+ concentrations were calculated by single exponential fitting of the kinetics data (as in panel C) and Km[Mg2+] values were calculated as described in ‘Materials and Methods’ section. (F) pH profiles of the reactions described in panel C in the absence and the presence of Gre. The values of kobs at different pH were calculated by single exponential fitting of the kinetics data (see panel C and ‘Materials and Methods’ section). Note that reactions with Gre were performed at 20°C (as in panel C).

Mentions: Taken together, the existing data indicate that the Mg2+ ions and the TL together form the active centre of RNAP. However, while the configuration of the aspartate triad remains constant, the TL may exist in a catalytically competent, folded state and in an inactive, unfolded state (4,5,7). In the unfolded state, the catalytic residues β′R1239 and β′H1242 are too far from the reactants to participate in catalysis (Figure 1A). Folding of the TL brings them to a catalytically active position (Figure 1A). Such an ability to bring and remove one of the two parts of the active centre may serve as a mechanism to provide fidelity for RNA synthesis (4), adaptation and possibly regulation of RNAP catalytic activities (13).Figure 1.


Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre.

Roghanian M, Yuzenkova Y, Zenkin N - Nucleic Acids Res. (2011)

(A) Upon folding the TL forms part of the RNAP active centre. The TL of T. thermophilus RNAP [PDB 2BE5 and 2O5J, respectively (7,34)], which has complete conservation with the active centre amino acids of T. aquaticus RNAP used in our study, in unfolded and folded states is coloured green and cyan, respectively. The amino acids of the TL that form the ‘TL active centre’ upon TL folding (M1238, R1239, H1242, T1243) are shown in space fill in both folded and unfolded states of the TL. The amino acids (not belonging to the TL) that surround catalytic Mg2+ ions and the incoming NTP are shown in grey spacefill. The aspartate triad is shown as purple sticks. Mg2+ ions of the active centre are shown as spheres. Incoming NTP, RNA 3′-end and DNA template bases are shown as orange, red and black sticks, respectively. The N-terminal coiled-coil domain of Gre factor (structure of E. coli GreA was used; PDB 1GRJ) which, according to our results, substitutes for the TL upon misincorporation or backtracking (see main text), was positioned to reach the active centre of RNAP without significant clashes with surrounding structure, and is shown as yellow ribbons. (B) Elongation complexes used in this study. Shown are the sequences of RNA, template strand (T DNA) and non-template strand (NT DNA) used to assemble elongation complexes (see ‘Materials and Methods’ section). (C) Gre-catalysed hydrolysis is independent of the TL. Kinetics of the cleavage reaction (also schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (red) and ΔTL RNAP (blue), in the absence (left) or presence (right) of Gre factor. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). The grey arrows represent the contributions of the TL and Gre to the rate of the reaction. Representative gels for cleavage by WT and ΔTL RNAPs in the absence or the presence of Gre are shown below the plots. Note that the kinetics of Gre catalysed reactions were measured at 20°C to reduce the rate of the reaction to allow it to be measured manually. (D) Gre-catalysed hydrolysis is independent of the H1242 of the TL. Kinetics of the cleavage reaction in mEC15 (as in panel C) by H1242A RNAP with and without Gre, and WT RNAP with Gre. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (E) The TL is not involved in Mg2+ chelation during phosphodiester bond hydrolysis. Dependence of kobs on the Mg2+ concentration during intrinsic cleavage (as in panel C) by WT (red) and ΔTL (blue) RNAPs. The values of apparent Km[Mg2+] are shown below the plots. The values of kobs at different Mg2+ concentrations were calculated by single exponential fitting of the kinetics data (as in panel C) and Km[Mg2+] values were calculated as described in ‘Materials and Methods’ section. (F) pH profiles of the reactions described in panel C in the absence and the presence of Gre. The values of kobs at different pH were calculated by single exponential fitting of the kinetics data (see panel C and ‘Materials and Methods’ section). Note that reactions with Gre were performed at 20°C (as in panel C).
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Figure 1: (A) Upon folding the TL forms part of the RNAP active centre. The TL of T. thermophilus RNAP [PDB 2BE5 and 2O5J, respectively (7,34)], which has complete conservation with the active centre amino acids of T. aquaticus RNAP used in our study, in unfolded and folded states is coloured green and cyan, respectively. The amino acids of the TL that form the ‘TL active centre’ upon TL folding (M1238, R1239, H1242, T1243) are shown in space fill in both folded and unfolded states of the TL. The amino acids (not belonging to the TL) that surround catalytic Mg2+ ions and the incoming NTP are shown in grey spacefill. The aspartate triad is shown as purple sticks. Mg2+ ions of the active centre are shown as spheres. Incoming NTP, RNA 3′-end and DNA template bases are shown as orange, red and black sticks, respectively. The N-terminal coiled-coil domain of Gre factor (structure of E. coli GreA was used; PDB 1GRJ) which, according to our results, substitutes for the TL upon misincorporation or backtracking (see main text), was positioned to reach the active centre of RNAP without significant clashes with surrounding structure, and is shown as yellow ribbons. (B) Elongation complexes used in this study. Shown are the sequences of RNA, template strand (T DNA) and non-template strand (NT DNA) used to assemble elongation complexes (see ‘Materials and Methods’ section). (C) Gre-catalysed hydrolysis is independent of the TL. Kinetics of the cleavage reaction (also schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (red) and ΔTL RNAP (blue), in the absence (left) or presence (right) of Gre factor. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). The grey arrows represent the contributions of the TL and Gre to the rate of the reaction. Representative gels for cleavage by WT and ΔTL RNAPs in the absence or the presence of Gre are shown below the plots. Note that the kinetics of Gre catalysed reactions were measured at 20°C to reduce the rate of the reaction to allow it to be measured manually. (D) Gre-catalysed hydrolysis is independent of the H1242 of the TL. Kinetics of the cleavage reaction in mEC15 (as in panel C) by H1242A RNAP with and without Gre, and WT RNAP with Gre. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (E) The TL is not involved in Mg2+ chelation during phosphodiester bond hydrolysis. Dependence of kobs on the Mg2+ concentration during intrinsic cleavage (as in panel C) by WT (red) and ΔTL (blue) RNAPs. The values of apparent Km[Mg2+] are shown below the plots. The values of kobs at different Mg2+ concentrations were calculated by single exponential fitting of the kinetics data (as in panel C) and Km[Mg2+] values were calculated as described in ‘Materials and Methods’ section. (F) pH profiles of the reactions described in panel C in the absence and the presence of Gre. The values of kobs at different pH were calculated by single exponential fitting of the kinetics data (see panel C and ‘Materials and Methods’ section). Note that reactions with Gre were performed at 20°C (as in panel C).
Mentions: Taken together, the existing data indicate that the Mg2+ ions and the TL together form the active centre of RNAP. However, while the configuration of the aspartate triad remains constant, the TL may exist in a catalytically competent, folded state and in an inactive, unfolded state (4,5,7). In the unfolded state, the catalytic residues β′R1239 and β′H1242 are too far from the reactants to participate in catalysis (Figure 1A). Folding of the TL brings them to a catalytically active position (Figure 1A). Such an ability to bring and remove one of the two parts of the active centre may serve as a mechanism to provide fidelity for RNA synthesis (4), adaptation and possibly regulation of RNAP catalytic activities (13).Figure 1.

Bottom Line: Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript.Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre.This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne NE2 4AX, UK.

ABSTRACT
The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

Show MeSH
Related in: MedlinePlus