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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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Role of Q1235 in discrimination against c2'dNTPs and c3'dNTPs and in catalysis. (a) Kinetics of incorporation of saturating cGTP (1 mM) in ECG1, c2'dATP (4 mM) and c3'dATP (1 mM) in ECA by wild-type (WT; red squares) and Q1235A (violet circles) RNAPs. Kinetic discrimination against c2'dATP and c3'dATP was quantified as a ratio of the rate of cGTP incorporation to the rate of incorporation of corresponding erroneous substrate. (b) Q1235 does not participate in catalysis directly. Kinetics of saturating (1 mM) cGTP incorporation in ECG1 by R1239A/H1242A (orange circles) and Q1235A/R1239A/H1242A (cyan triangles) RNA polymerase. Compare to panel A (left plot) and to Figure 3e.
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Figure 4: Role of Q1235 in discrimination against c2'dNTPs and c3'dNTPs and in catalysis. (a) Kinetics of incorporation of saturating cGTP (1 mM) in ECG1, c2'dATP (4 mM) and c3'dATP (1 mM) in ECA by wild-type (WT; red squares) and Q1235A (violet circles) RNAPs. Kinetic discrimination against c2'dATP and c3'dATP was quantified as a ratio of the rate of cGTP incorporation to the rate of incorporation of corresponding erroneous substrate. (b) Q1235 does not participate in catalysis directly. Kinetics of saturating (1 mM) cGTP incorporation in ECG1 by R1239A/H1242A (orange circles) and Q1235A/R1239A/H1242A (cyan triangles) RNA polymerase. Compare to panel A (left plot) and to Figure 3e.

Mentions: The similarity of the effects caused by individual R1239A and H1242A substitutions on phosphotransfer reactions disfavours the exclusive role of H1242 in catalysis as a Brønsted-Lowry or Lewis acid [11,12]. Nevertheless, we measured pH dependences of catalytic rates of WT, R1239A, H1242A and R1239A/H1242A RNAPs. We used 1 mM cNTP to avoid effects of pH on Kd [cNTP]. The shapes of curves for all four RNAPs were the same (Figure 4d). The descending limb that corresponds to protonation of the leaving group was not observed in either of the plots. Inflection at pH = ~8.5-9.2 can be assigned to pKa of deprotonation of RNA 3' hydroxyl - that is, of a general base. Note that the substitution of either R1239 or H1242 shifts this pKa towards a more basic value, though by less than one pH unit (Figure 4d). Therefore, H1242 and R1239 participate in general base catalysis (in contrast to earlier proposed role in general acid catalysis [12]), but are likely do so as a part of a network of other groups of the active centre. This view is supported by the fact that the slopes of log-log curves were less than 1 indicating that the inflection point does not reflect a single pKa that is being titrated. Furthermore, substitution of R1239 to asparagine (naturally present in this position in eukaryotic RNAP II) only slightly affected catalysis (Figure 4e), although the side chains of these amino acids are of different chemical nature.


Stepwise mechanism for transcription fidelity.

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

Role of Q1235 in discrimination against c2'dNTPs and c3'dNTPs and in catalysis. (a) Kinetics of incorporation of saturating cGTP (1 mM) in ECG1, c2'dATP (4 mM) and c3'dATP (1 mM) in ECA by wild-type (WT; red squares) and Q1235A (violet circles) RNAPs. Kinetic discrimination against c2'dATP and c3'dATP was quantified as a ratio of the rate of cGTP incorporation to the rate of incorporation of corresponding erroneous substrate. (b) Q1235 does not participate in catalysis directly. Kinetics of saturating (1 mM) cGTP incorporation in ECG1 by R1239A/H1242A (orange circles) and Q1235A/R1239A/H1242A (cyan triangles) RNA polymerase. Compare to panel A (left plot) and to Figure 3e.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874521&req=5

Figure 4: Role of Q1235 in discrimination against c2'dNTPs and c3'dNTPs and in catalysis. (a) Kinetics of incorporation of saturating cGTP (1 mM) in ECG1, c2'dATP (4 mM) and c3'dATP (1 mM) in ECA by wild-type (WT; red squares) and Q1235A (violet circles) RNAPs. Kinetic discrimination against c2'dATP and c3'dATP was quantified as a ratio of the rate of cGTP incorporation to the rate of incorporation of corresponding erroneous substrate. (b) Q1235 does not participate in catalysis directly. Kinetics of saturating (1 mM) cGTP incorporation in ECG1 by R1239A/H1242A (orange circles) and Q1235A/R1239A/H1242A (cyan triangles) RNA polymerase. Compare to panel A (left plot) and to Figure 3e.
Mentions: The similarity of the effects caused by individual R1239A and H1242A substitutions on phosphotransfer reactions disfavours the exclusive role of H1242 in catalysis as a Brønsted-Lowry or Lewis acid [11,12]. Nevertheless, we measured pH dependences of catalytic rates of WT, R1239A, H1242A and R1239A/H1242A RNAPs. We used 1 mM cNTP to avoid effects of pH on Kd [cNTP]. The shapes of curves for all four RNAPs were the same (Figure 4d). The descending limb that corresponds to protonation of the leaving group was not observed in either of the plots. Inflection at pH = ~8.5-9.2 can be assigned to pKa of deprotonation of RNA 3' hydroxyl - that is, of a general base. Note that the substitution of either R1239 or H1242 shifts this pKa towards a more basic value, though by less than one pH unit (Figure 4d). Therefore, H1242 and R1239 participate in general base catalysis (in contrast to earlier proposed role in general acid catalysis [12]), but are likely do so as a part of a network of other groups of the active centre. This view is supported by the fact that the slopes of log-log curves were less than 1 indicating that the inflection point does not reflect a single pKa that is being titrated. Furthermore, substitution of R1239 to asparagine (naturally present in this position in eukaryotic RNAP II) only slightly affected catalysis (Figure 4e), although the side chains of these amino acids are of different chemical nature.

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