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Stepwise mechanism for transcription fidelity.

Yuzenkova Y, Bochkareva A, Tadigotla VR, Roghanian M, Zorov S, Severinov K, Zenkin N - BMC Biol. (2010)

Bottom Line: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process.We show that individual steps contribute differently to discrimination against various erroneous substrates.We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4AX, UK.

ABSTRACT

Background: Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.

Results: Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH.

Conclusions: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

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Roles of TL amino acids in catalysis. (a). Reaction rates in saturating (1 mM) cGTP in ECG1 and Kd [ncGTP] by wild-type (WT), M1238A, M1238V and M1238L are shown below the cartoons of the active centres of the corresponding enzymes, drawn in PyMol using PDB 2O5J and 'mutagenesis' function (colour code as in Figure 1). (b) Kinetics of pyrophosphorolysis by WT, R1239A and H1242A in the presence of 0.5 mM PPi in ECG1 (Additional File 1: Figure S2) with 32P 5'-labelled RNA that was walked by two positions (G and A). (c) Kinetics of intrinsic transcript hydrolysis by WT (red squares), Q1235A (violet circles) and R1239A (green circles) RNA polymerase (RNAP) in EChydr (Additional File 1: Figure S2) with 32P 5'-labelled RNA. The lines in the plot are the non-linear regression fits of the data. (d) pH dependences of the rates of 1 mM cGTP incorporation in ECG1 at 20°C by WT, R1239A, H1242A and R1239A/H1239A RNAPs. Solid lines show fits of the data to a sigmoidal function, and pKa values retrieved from these fits are shown above the plots. (e) Kinetics of 1 μM cGTP incorporation in ECG1 by WT (red squares), R1239A (green circles) and R1239N (blue triangles). Solid lines show fits of the data to an exponential function.
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Figure 3: Roles of TL amino acids in catalysis. (a). Reaction rates in saturating (1 mM) cGTP in ECG1 and Kd [ncGTP] by wild-type (WT), M1238A, M1238V and M1238L are shown below the cartoons of the active centres of the corresponding enzymes, drawn in PyMol using PDB 2O5J and 'mutagenesis' function (colour code as in Figure 1). (b) Kinetics of pyrophosphorolysis by WT, R1239A and H1242A in the presence of 0.5 mM PPi in ECG1 (Additional File 1: Figure S2) with 32P 5'-labelled RNA that was walked by two positions (G and A). (c) Kinetics of intrinsic transcript hydrolysis by WT (red squares), Q1235A (violet circles) and R1239A (green circles) RNA polymerase (RNAP) in EChydr (Additional File 1: Figure S2) with 32P 5'-labelled RNA. The lines in the plot are the non-linear regression fits of the data. (d) pH dependences of the rates of 1 mM cGTP incorporation in ECG1 at 20°C by WT, R1239A, H1242A and R1239A/H1239A RNAPs. Solid lines show fits of the data to a sigmoidal function, and pKa values retrieved from these fits are shown above the plots. (e) Kinetics of 1 μM cGTP incorporation in ECG1 by WT (red squares), R1239A (green circles) and R1239N (blue triangles). Solid lines show fits of the data to an exponential function.

Mentions: In order to further investigate the competition between folded TL and ncNTP bound in the active centre, we analysed Kd [ncNTP] for RNAPs bearing single substitutions in TL. From the mutant enzymes tested (Table 2) only M1238A RNAP had Kd [ncNTP] that was significantly lower than the WT RNAP value and close to that of ΔTL RNAP (Table 1). This suggests that M1238 is required for ncNTP expulsion from the active centre. In the folded TL, M1238 stacks on the base of cNTP in the i+1 site (Figure 1, 3a). We hypothesized that M1238 is required for stabilization of the TL in the folded conformation via interaction with the incoming substrate base and, thus, is responsible for the observed competition between folded TL and ncNTP. To test this notion, we analysed RNAPs with substitutions of M1238 to hydrophobic amino acids with larger than alanine side chains, M1238V and M1238L (Figure 3a). In support of our hypothesis, M1238V RNAP had Kd [ncNTP] = 1700 ± 500 μM (that is, intermediate between those of M1238A and WT RNAPs) while M1238L RNAP had the same Kd [ncNTP] = 2800 ± 150 μM as WT RNAP (Figure 3a).


Stepwise mechanism for transcription fidelity.

Yuzenkova Y, Bochkareva A, Tadigotla VR, Roghanian M, Zorov S, Severinov K, Zenkin N - BMC Biol. (2010)

Roles of TL amino acids in catalysis. (a). Reaction rates in saturating (1 mM) cGTP in ECG1 and Kd [ncGTP] by wild-type (WT), M1238A, M1238V and M1238L are shown below the cartoons of the active centres of the corresponding enzymes, drawn in PyMol using PDB 2O5J and 'mutagenesis' function (colour code as in Figure 1). (b) Kinetics of pyrophosphorolysis by WT, R1239A and H1242A in the presence of 0.5 mM PPi in ECG1 (Additional File 1: Figure S2) with 32P 5'-labelled RNA that was walked by two positions (G and A). (c) Kinetics of intrinsic transcript hydrolysis by WT (red squares), Q1235A (violet circles) and R1239A (green circles) RNA polymerase (RNAP) in EChydr (Additional File 1: Figure S2) with 32P 5'-labelled RNA. The lines in the plot are the non-linear regression fits of the data. (d) pH dependences of the rates of 1 mM cGTP incorporation in ECG1 at 20°C by WT, R1239A, H1242A and R1239A/H1239A RNAPs. Solid lines show fits of the data to a sigmoidal function, and pKa values retrieved from these fits are shown above the plots. (e) Kinetics of 1 μM cGTP incorporation in ECG1 by WT (red squares), R1239A (green circles) and R1239N (blue triangles). Solid lines show fits of the data to an exponential function.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Roles of TL amino acids in catalysis. (a). Reaction rates in saturating (1 mM) cGTP in ECG1 and Kd [ncGTP] by wild-type (WT), M1238A, M1238V and M1238L are shown below the cartoons of the active centres of the corresponding enzymes, drawn in PyMol using PDB 2O5J and 'mutagenesis' function (colour code as in Figure 1). (b) Kinetics of pyrophosphorolysis by WT, R1239A and H1242A in the presence of 0.5 mM PPi in ECG1 (Additional File 1: Figure S2) with 32P 5'-labelled RNA that was walked by two positions (G and A). (c) Kinetics of intrinsic transcript hydrolysis by WT (red squares), Q1235A (violet circles) and R1239A (green circles) RNA polymerase (RNAP) in EChydr (Additional File 1: Figure S2) with 32P 5'-labelled RNA. The lines in the plot are the non-linear regression fits of the data. (d) pH dependences of the rates of 1 mM cGTP incorporation in ECG1 at 20°C by WT, R1239A, H1242A and R1239A/H1239A RNAPs. Solid lines show fits of the data to a sigmoidal function, and pKa values retrieved from these fits are shown above the plots. (e) Kinetics of 1 μM cGTP incorporation in ECG1 by WT (red squares), R1239A (green circles) and R1239N (blue triangles). Solid lines show fits of the data to an exponential function.
Mentions: In order to further investigate the competition between folded TL and ncNTP bound in the active centre, we analysed Kd [ncNTP] for RNAPs bearing single substitutions in TL. From the mutant enzymes tested (Table 2) only M1238A RNAP had Kd [ncNTP] that was significantly lower than the WT RNAP value and close to that of ΔTL RNAP (Table 1). This suggests that M1238 is required for ncNTP expulsion from the active centre. In the folded TL, M1238 stacks on the base of cNTP in the i+1 site (Figure 1, 3a). We hypothesized that M1238 is required for stabilization of the TL in the folded conformation via interaction with the incoming substrate base and, thus, is responsible for the observed competition between folded TL and ncNTP. To test this notion, we analysed RNAPs with substitutions of M1238 to hydrophobic amino acids with larger than alanine side chains, M1238V and M1238L (Figure 3a). In support of our hypothesis, M1238V RNAP had Kd [ncNTP] = 1700 ± 500 μM (that is, intermediate between those of M1238A and WT RNAPs) while M1238L RNAP had the same Kd [ncNTP] = 2800 ± 150 μM as WT RNAP (Figure 3a).

Bottom Line: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process.We show that individual steps contribute differently to discrimination against various erroneous substrates.We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4AX, UK.

ABSTRACT

Background: Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.

Results: Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH.

Conclusions: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

Show MeSH
Related in: MedlinePlus