Limits...
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.

Show MeSH

Related in: MedlinePlus

Two models of switching of the active centres, the ‘TL active centre’ (red) and ‘Gre active centre’ (blue), in the RNA polymerase catalytic site (yellow circle). Gre does not interfere with the TL during productive elongation. However, in response to misincorporation or occasional backtracking Gre substitutes for the TL in the active site where it activates the hydrolytic activity to efficiently resolve backtracked complexes. After resolution of backtracked complex is accomplished, Gre is replaced back by the TL allowing continuation of elongation. Switching may take place without (A) or with (B) dissociation of Gre from the elongation complex.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105419&req=5

Figure 3: Two models of switching of the active centres, the ‘TL active centre’ (red) and ‘Gre active centre’ (blue), in the RNA polymerase catalytic site (yellow circle). Gre does not interfere with the TL during productive elongation. However, in response to misincorporation or occasional backtracking Gre substitutes for the TL in the active site where it activates the hydrolytic activity to efficiently resolve backtracked complexes. After resolution of backtracked complex is accomplished, Gre is replaced back by the TL allowing continuation of elongation. Switching may take place without (A) or with (B) dissociation of Gre from the elongation complex.

Mentions: The above results suggest that, while acting in the active centre, Gre should switch off the TL catalysed cleavage. This may happen through physical blocking of TL folding upon Gre binding in the secondary channel. To test this hypothesis we used a mutant Gre factor that had two of the residues essential for catalysis [D42 and E45 (17,18)] changed to alanines, GreD42A/E45A. As seen from Figure 2A, addition of GreD42A/E45A strongly inhibited the intrinsic cleavage (∼30-fold). Cleavage by ΔTL RNAP was not affected by GreD42A/E45A (Figure 2A). We cannot exclude the possibility that the deletion of the TL affects binding of GreD42A/E45A to ΔTL RNAP. However, only slightly unaltered activity of the wild-type Gre with ΔTL RNAP argues that binding of the mutant Gre to ΔTL RNAP in a functional conformation is not affected significantly (see previous section). The results, therefore, indicate that, upon binding, Gre factor indeed inactivates the TL and thus turns off the intrinsic cleavage activity of the RNAP. The lack of full inactivation of TL dependent hydrolysis by GreD42A/E45A (to the level of hydrolysis rate by ΔTL RNAP) can be explained by temporary dissociation of GreD42A/E45A from the elongation complex during long incubations, which presumably allows the TL to assist hydrolysis. Since the folded TL, along with Mg2+ ions, forms the active centre of RNAP, Gre substitution of the TL leads to exchange of the amino acid content and, as a result, of the catalytic properties of RNAP active centre. Therefore, this can be considered a substitution of the active centres of RNAP (see schemes in Figures 1A and 3).Figure 2.


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

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

Two models of switching of the active centres, the ‘TL active centre’ (red) and ‘Gre active centre’ (blue), in the RNA polymerase catalytic site (yellow circle). Gre does not interfere with the TL during productive elongation. However, in response to misincorporation or occasional backtracking Gre substitutes for the TL in the active site where it activates the hydrolytic activity to efficiently resolve backtracked complexes. After resolution of backtracked complex is accomplished, Gre is replaced back by the TL allowing continuation of elongation. Switching may take place without (A) or with (B) dissociation of Gre from the elongation complex.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105419&req=5

Figure 3: Two models of switching of the active centres, the ‘TL active centre’ (red) and ‘Gre active centre’ (blue), in the RNA polymerase catalytic site (yellow circle). Gre does not interfere with the TL during productive elongation. However, in response to misincorporation or occasional backtracking Gre substitutes for the TL in the active site where it activates the hydrolytic activity to efficiently resolve backtracked complexes. After resolution of backtracked complex is accomplished, Gre is replaced back by the TL allowing continuation of elongation. Switching may take place without (A) or with (B) dissociation of Gre from the elongation complex.
Mentions: The above results suggest that, while acting in the active centre, Gre should switch off the TL catalysed cleavage. This may happen through physical blocking of TL folding upon Gre binding in the secondary channel. To test this hypothesis we used a mutant Gre factor that had two of the residues essential for catalysis [D42 and E45 (17,18)] changed to alanines, GreD42A/E45A. As seen from Figure 2A, addition of GreD42A/E45A strongly inhibited the intrinsic cleavage (∼30-fold). Cleavage by ΔTL RNAP was not affected by GreD42A/E45A (Figure 2A). We cannot exclude the possibility that the deletion of the TL affects binding of GreD42A/E45A to ΔTL RNAP. However, only slightly unaltered activity of the wild-type Gre with ΔTL RNAP argues that binding of the mutant Gre to ΔTL RNAP in a functional conformation is not affected significantly (see previous section). The results, therefore, indicate that, upon binding, Gre factor indeed inactivates the TL and thus turns off the intrinsic cleavage activity of the RNAP. The lack of full inactivation of TL dependent hydrolysis by GreD42A/E45A (to the level of hydrolysis rate by ΔTL RNAP) can be explained by temporary dissociation of GreD42A/E45A from the elongation complex during long incubations, which presumably allows the TL to assist hydrolysis. Since the folded TL, along with Mg2+ ions, forms the active centre of RNAP, Gre substitution of the TL leads to exchange of the amino acid content and, as a result, of the catalytic properties of RNAP active centre. Therefore, this can be considered a substitution of the active centres of RNAP (see schemes in Figures 1A and 3).Figure 2.

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