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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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Related in: MedlinePlus

Incorporation and misincorporation by wild-type (WT) and mutant RNA polymerase (RNAPs). (a) Cartoon schematically describes the reaction of cNTP (cGTP) incorporation in ECG1 (Additional File1: Figure S2) with 32P 5'-labelled RNA (asterisk). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Representative gels of 100 μM cGTP incorporation by WT and 500 μM cGTP incorporation by ΔTL in ECG1 are shown. The lack of complete extension of transcripts was due to the procedure by which elongation complexes were assembled (see Methods). (b) The intrinsic proofreading reaction accompanies misincorporation. The cartoon above the gel schematically describes the processes going on during ncGTP misincorporation in ECA (Additional File 1: Figure S2). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Note that RNAs in elongation complexes were labelled at the 3' end (by incorporation of [α32P]GTP; asterisk), thus allowing monitoring both misincorporation event and removal of the wrong nucleotide via transcript assisted proofreading. Misincorporation of 1 mM ncGTP and proofreading by WT, H1242A and R1239A RNAPs are shown as an example. The cleavage products larger than dinucleotide originate from 2 bp and 3 bp backtracked complexes that undergone further extension after misincorporation. The colours of the RNA products of the reactions are the same as in the scheme of the reaction above the gels. Black vertical line separates lanes originating from the same gel that were brought together.
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Figure 2: Incorporation and misincorporation by wild-type (WT) and mutant RNA polymerase (RNAPs). (a) Cartoon schematically describes the reaction of cNTP (cGTP) incorporation in ECG1 (Additional File1: Figure S2) with 32P 5'-labelled RNA (asterisk). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Representative gels of 100 μM cGTP incorporation by WT and 500 μM cGTP incorporation by ΔTL in ECG1 are shown. The lack of complete extension of transcripts was due to the procedure by which elongation complexes were assembled (see Methods). (b) The intrinsic proofreading reaction accompanies misincorporation. The cartoon above the gel schematically describes the processes going on during ncGTP misincorporation in ECA (Additional File 1: Figure S2). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Note that RNAs in elongation complexes were labelled at the 3' end (by incorporation of [α32P]GTP; asterisk), thus allowing monitoring both misincorporation event and removal of the wrong nucleotide via transcript assisted proofreading. Misincorporation of 1 mM ncGTP and proofreading by WT, H1242A and R1239A RNAPs are shown as an example. The cleavage products larger than dinucleotide originate from 2 bp and 3 bp backtracked complexes that undergone further extension after misincorporation. The colours of the RNA products of the reactions are the same as in the scheme of the reaction above the gels. Black vertical line separates lanes originating from the same gel that were brought together.

Mentions: For the remaining mutants, we determined kpol (catalytic rate at saturating substrate concentration) and Kd (substrate dissociation constant) of single nucleotide incorporation and misincorporation reactions using artificially assembled elongation complexes [7,15,16] (Figure 2, Additional File 1: Figure S2). The kinetic analysis of the data was performed as described in Materials and Methods and Additional File 1: Supplementary Methods.


Stepwise mechanism for transcription fidelity.

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

Incorporation and misincorporation by wild-type (WT) and mutant RNA polymerase (RNAPs). (a) Cartoon schematically describes the reaction of cNTP (cGTP) incorporation in ECG1 (Additional File1: Figure S2) with 32P 5'-labelled RNA (asterisk). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Representative gels of 100 μM cGTP incorporation by WT and 500 μM cGTP incorporation by ΔTL in ECG1 are shown. The lack of complete extension of transcripts was due to the procedure by which elongation complexes were assembled (see Methods). (b) The intrinsic proofreading reaction accompanies misincorporation. The cartoon above the gel schematically describes the processes going on during ncGTP misincorporation in ECA (Additional File 1: Figure S2). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Note that RNAs in elongation complexes were labelled at the 3' end (by incorporation of [α32P]GTP; asterisk), thus allowing monitoring both misincorporation event and removal of the wrong nucleotide via transcript assisted proofreading. Misincorporation of 1 mM ncGTP and proofreading by WT, H1242A and R1239A RNAPs are shown as an example. The cleavage products larger than dinucleotide originate from 2 bp and 3 bp backtracked complexes that undergone further extension after misincorporation. The colours of the RNA products of the reactions are the same as in the scheme of the reaction above the gels. Black vertical line separates lanes originating from the same gel that were brought together.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Incorporation and misincorporation by wild-type (WT) and mutant RNA polymerase (RNAPs). (a) Cartoon schematically describes the reaction of cNTP (cGTP) incorporation in ECG1 (Additional File1: Figure S2) with 32P 5'-labelled RNA (asterisk). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Representative gels of 100 μM cGTP incorporation by WT and 500 μM cGTP incorporation by ΔTL in ECG1 are shown. The lack of complete extension of transcripts was due to the procedure by which elongation complexes were assembled (see Methods). (b) The intrinsic proofreading reaction accompanies misincorporation. The cartoon above the gel schematically describes the processes going on during ncGTP misincorporation in ECA (Additional File 1: Figure S2). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Note that RNAs in elongation complexes were labelled at the 3' end (by incorporation of [α32P]GTP; asterisk), thus allowing monitoring both misincorporation event and removal of the wrong nucleotide via transcript assisted proofreading. Misincorporation of 1 mM ncGTP and proofreading by WT, H1242A and R1239A RNAPs are shown as an example. The cleavage products larger than dinucleotide originate from 2 bp and 3 bp backtracked complexes that undergone further extension after misincorporation. The colours of the RNA products of the reactions are the same as in the scheme of the reaction above the gels. Black vertical line separates lanes originating from the same gel that were brought together.
Mentions: For the remaining mutants, we determined kpol (catalytic rate at saturating substrate concentration) and Kd (substrate dissociation constant) of single nucleotide incorporation and misincorporation reactions using artificially assembled elongation complexes [7,15,16] (Figure 2, Additional File 1: Figure S2). The kinetic analysis of the data was performed as described in Materials and Methods and Additional File 1: Supplementary Methods.

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