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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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Substitution of the TL by Gre depends on the state of the elongation complex. (A) Kinetics of the cleavage reaction (schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (left) and ΔTL RNAP (right) in the absence (red) or presence (blue) of the catalytically inactive mutant Gre factor, GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (B) Kinetics of single nucleotide addition (1 µM and 1 mM CTP; reaction schematically shown above the plots) in cEC15 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of GreD42A/E45A. Solid curves are the single (without GreD42A/E45A) or double (with GreD42A/E45A) exponential fits of the kinetics data (see ‘Materials and Methods’ section). Blue vertical lines in the left plot show the inhibited (slow) and non-inhibited (fast) fractions of elongation complexes. (C) Kinetics of pyrophosphorolysis and second phosphodiester bond hydrolysis in cEC13 and cEC15. RNAs longer than 13 nt in lanes 2–4 originate from incorporation of NTPs, which are the result of pyrophosphorolysis. (D) Exo III footprinting of the front edge of RNAP in cEC13 and cEC15. Non-template DNA strand (NT DNA) and RNA were labelled with P32 at the 5′-end (asterisks in the scheme on the left). Three different concentrations of Exo III were used for accurate comparison of relative distribution of translocation states in cEC13 and cEC15. The lower panel originates from the same gel as the top panel, and shows the transcripts in the complexes. Black vertical lines in both panels separate lanes originating from the same gel that were brought together. (E) Kinetics of single nucleotide addition (1 µM GTP, reaction schematically shown above the plots) in cEC13 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of the GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section).
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Figure 2: Substitution of the TL by Gre depends on the state of the elongation complex. (A) Kinetics of the cleavage reaction (schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (left) and ΔTL RNAP (right) in the absence (red) or presence (blue) of the catalytically inactive mutant Gre factor, GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (B) Kinetics of single nucleotide addition (1 µM and 1 mM CTP; reaction schematically shown above the plots) in cEC15 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of GreD42A/E45A. Solid curves are the single (without GreD42A/E45A) or double (with GreD42A/E45A) exponential fits of the kinetics data (see ‘Materials and Methods’ section). Blue vertical lines in the left plot show the inhibited (slow) and non-inhibited (fast) fractions of elongation complexes. (C) Kinetics of pyrophosphorolysis and second phosphodiester bond hydrolysis in cEC13 and cEC15. RNAs longer than 13 nt in lanes 2–4 originate from incorporation of NTPs, which are the result of pyrophosphorolysis. (D) Exo III footprinting of the front edge of RNAP in cEC13 and cEC15. Non-template DNA strand (NT DNA) and RNA were labelled with P32 at the 5′-end (asterisks in the scheme on the left). Three different concentrations of Exo III were used for accurate comparison of relative distribution of translocation states in cEC13 and cEC15. The lower panel originates from the same gel as the top panel, and shows the transcripts in the complexes. Black vertical lines in both panels separate lanes originating from the same gel that were brought together. (E) Kinetics of single nucleotide addition (1 µM GTP, reaction schematically shown above the plots) in cEC13 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of the GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section).

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)

Substitution of the TL by Gre depends on the state of the elongation complex. (A) Kinetics of the cleavage reaction (schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (left) and ΔTL RNAP (right) in the absence (red) or presence (blue) of the catalytically inactive mutant Gre factor, GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (B) Kinetics of single nucleotide addition (1 µM and 1 mM CTP; reaction schematically shown above the plots) in cEC15 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of GreD42A/E45A. Solid curves are the single (without GreD42A/E45A) or double (with GreD42A/E45A) exponential fits of the kinetics data (see ‘Materials and Methods’ section). Blue vertical lines in the left plot show the inhibited (slow) and non-inhibited (fast) fractions of elongation complexes. (C) Kinetics of pyrophosphorolysis and second phosphodiester bond hydrolysis in cEC13 and cEC15. RNAs longer than 13 nt in lanes 2–4 originate from incorporation of NTPs, which are the result of pyrophosphorolysis. (D) Exo III footprinting of the front edge of RNAP in cEC13 and cEC15. Non-template DNA strand (NT DNA) and RNA were labelled with P32 at the 5′-end (asterisks in the scheme on the left). Three different concentrations of Exo III were used for accurate comparison of relative distribution of translocation states in cEC13 and cEC15. The lower panel originates from the same gel as the top panel, and shows the transcripts in the complexes. Black vertical lines in both panels separate lanes originating from the same gel that were brought together. (E) Kinetics of single nucleotide addition (1 µM GTP, reaction schematically shown above the plots) in cEC13 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of the GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section).
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Figure 2: Substitution of the TL by Gre depends on the state of the elongation complex. (A) Kinetics of the cleavage reaction (schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (left) and ΔTL RNAP (right) in the absence (red) or presence (blue) of the catalytically inactive mutant Gre factor, GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (B) Kinetics of single nucleotide addition (1 µM and 1 mM CTP; reaction schematically shown above the plots) in cEC15 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of GreD42A/E45A. Solid curves are the single (without GreD42A/E45A) or double (with GreD42A/E45A) exponential fits of the kinetics data (see ‘Materials and Methods’ section). Blue vertical lines in the left plot show the inhibited (slow) and non-inhibited (fast) fractions of elongation complexes. (C) Kinetics of pyrophosphorolysis and second phosphodiester bond hydrolysis in cEC13 and cEC15. RNAs longer than 13 nt in lanes 2–4 originate from incorporation of NTPs, which are the result of pyrophosphorolysis. (D) Exo III footprinting of the front edge of RNAP in cEC13 and cEC15. Non-template DNA strand (NT DNA) and RNA were labelled with P32 at the 5′-end (asterisks in the scheme on the left). Three different concentrations of Exo III were used for accurate comparison of relative distribution of translocation states in cEC13 and cEC15. The lower panel originates from the same gel as the top panel, and shows the transcripts in the complexes. Black vertical lines in both panels separate lanes originating from the same gel that were brought together. (E) Kinetics of single nucleotide addition (1 µM GTP, reaction schematically shown above the plots) in cEC13 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of the GreD42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section).
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