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Tagetitoxin inhibits transcription by stabilizing pre-translocated state of the elongation complex.

Yuzenkova Y, Roghanian M, Bochkareva A, Zenkin N - Nucleic Acids Res. (2013)

Bottom Line: We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex.The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex.Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

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
Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

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TGT targets the pre-translocated state of elongation complex. (A) Kinetics of NTP (1 µM) incorporation, pyrophosphorolysis (500 µM PPi) and phosphodiester bond hydrolysis in sECs and rECs. Representative gels are shown. (B) Summary of results on inhibition by TGT and the rates of reactions (kobs) in sECs and rECs (numbers that follow the ± sign are standard errors). Shades of gray in the ‘heat map’ reflect magnitude of effects: darkest gray corresponds to strongest inhibition of NTP addition by TGT or highest rates of reactions in the absence of TGT. Note the correlation between extent of inhibition of NTP addition by TGT and the rate of pyrophosphorolysis. The right column roughly shows the distribution between translocation states in elongation complexes, deduced from the rates of reactions. (C) Kinetics of NTP (1 µM) incorporation in the presence or absence of TGT in elongation complexes stabilized in the post-translocated (postEC14) and the pre-translocated (preEC15) states (12). Extent of inhibition by TGT is shown below gels.
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gkt708-F2: TGT targets the pre-translocated state of elongation complex. (A) Kinetics of NTP (1 µM) incorporation, pyrophosphorolysis (500 µM PPi) and phosphodiester bond hydrolysis in sECs and rECs. Representative gels are shown. (B) Summary of results on inhibition by TGT and the rates of reactions (kobs) in sECs and rECs (numbers that follow the ± sign are standard errors). Shades of gray in the ‘heat map’ reflect magnitude of effects: darkest gray corresponds to strongest inhibition of NTP addition by TGT or highest rates of reactions in the absence of TGT. Note the correlation between extent of inhibition of NTP addition by TGT and the rate of pyrophosphorolysis. The right column roughly shows the distribution between translocation states in elongation complexes, deduced from the rates of reactions. (C) Kinetics of NTP (1 µM) incorporation in the presence or absence of TGT in elongation complexes stabilized in the post-translocated (postEC14) and the pre-translocated (preEC15) states (12). Extent of inhibition by TGT is shown below gels.

Mentions: Inhibition by TGT depends on the transcribed sequence. (A) Scheme of the NAC and translocation oscillation of the elongation complex between post-translocated, pre-translocated and backtracked states. The states of the TL (folded-vertical versus unfolded-horizontal) are shown based on biochemical and crystallographic data, except for pre-translocated state, conformation of the TL in which is not known. Note the reactions that are catalyzed in each translocation state. (B) Transcription in the absence or presence of TGT in elongation complexes of unrelated transcribed sequences (t1 and t2, shown above the gels; RNA was labeled at the 5′-end). Depicted are sECs and rECs analyzed in our study. (C) Kinetics of NTP incorporation in sECs and rECs in the presence or absence of TGT. Observed rate constants (kobs) are shown below gels (numbers that follow the ± sign are standard errors). Note that division of complexes into sECs and rECs is formal, and a broad distribution of TGT sensitivity is observed (see also Figure 2B). (D) Characteristics of TGT action analyzed in sEC14t2 (scheme at the top; radiolabel is in bold): inhibition of incorporation of 1 µM and 1 mM NTP in the absence or presence of TGT (error bars are standard deviations); Ki[TGT] in the presence of 1 µM and 1 mM NTP; affinity and rate constants for NTP incorporation and pyrophosphorolysis in the absence and presence of TGT (numbers that follow the ± sign are standard errors). (E) Inhibition constants for two complexes with different extent of TGT inhibition (error bars are standard deviations, numbers that follow the ± sign are standard errors).


Tagetitoxin inhibits transcription by stabilizing pre-translocated state of the elongation complex.

Yuzenkova Y, Roghanian M, Bochkareva A, Zenkin N - Nucleic Acids Res. (2013)

TGT targets the pre-translocated state of elongation complex. (A) Kinetics of NTP (1 µM) incorporation, pyrophosphorolysis (500 µM PPi) and phosphodiester bond hydrolysis in sECs and rECs. Representative gels are shown. (B) Summary of results on inhibition by TGT and the rates of reactions (kobs) in sECs and rECs (numbers that follow the ± sign are standard errors). Shades of gray in the ‘heat map’ reflect magnitude of effects: darkest gray corresponds to strongest inhibition of NTP addition by TGT or highest rates of reactions in the absence of TGT. Note the correlation between extent of inhibition of NTP addition by TGT and the rate of pyrophosphorolysis. The right column roughly shows the distribution between translocation states in elongation complexes, deduced from the rates of reactions. (C) Kinetics of NTP (1 µM) incorporation in the presence or absence of TGT in elongation complexes stabilized in the post-translocated (postEC14) and the pre-translocated (preEC15) states (12). Extent of inhibition by TGT is shown below gels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814378&req=5

gkt708-F2: TGT targets the pre-translocated state of elongation complex. (A) Kinetics of NTP (1 µM) incorporation, pyrophosphorolysis (500 µM PPi) and phosphodiester bond hydrolysis in sECs and rECs. Representative gels are shown. (B) Summary of results on inhibition by TGT and the rates of reactions (kobs) in sECs and rECs (numbers that follow the ± sign are standard errors). Shades of gray in the ‘heat map’ reflect magnitude of effects: darkest gray corresponds to strongest inhibition of NTP addition by TGT or highest rates of reactions in the absence of TGT. Note the correlation between extent of inhibition of NTP addition by TGT and the rate of pyrophosphorolysis. The right column roughly shows the distribution between translocation states in elongation complexes, deduced from the rates of reactions. (C) Kinetics of NTP (1 µM) incorporation in the presence or absence of TGT in elongation complexes stabilized in the post-translocated (postEC14) and the pre-translocated (preEC15) states (12). Extent of inhibition by TGT is shown below gels.
Mentions: Inhibition by TGT depends on the transcribed sequence. (A) Scheme of the NAC and translocation oscillation of the elongation complex between post-translocated, pre-translocated and backtracked states. The states of the TL (folded-vertical versus unfolded-horizontal) are shown based on biochemical and crystallographic data, except for pre-translocated state, conformation of the TL in which is not known. Note the reactions that are catalyzed in each translocation state. (B) Transcription in the absence or presence of TGT in elongation complexes of unrelated transcribed sequences (t1 and t2, shown above the gels; RNA was labeled at the 5′-end). Depicted are sECs and rECs analyzed in our study. (C) Kinetics of NTP incorporation in sECs and rECs in the presence or absence of TGT. Observed rate constants (kobs) are shown below gels (numbers that follow the ± sign are standard errors). Note that division of complexes into sECs and rECs is formal, and a broad distribution of TGT sensitivity is observed (see also Figure 2B). (D) Characteristics of TGT action analyzed in sEC14t2 (scheme at the top; radiolabel is in bold): inhibition of incorporation of 1 µM and 1 mM NTP in the absence or presence of TGT (error bars are standard deviations); Ki[TGT] in the presence of 1 µM and 1 mM NTP; affinity and rate constants for NTP incorporation and pyrophosphorolysis in the absence and presence of TGT (numbers that follow the ± sign are standard errors). (E) Inhibition constants for two complexes with different extent of TGT inhibition (error bars are standard deviations, numbers that follow the ± sign are standard errors).

Bottom Line: We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex.The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex.Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

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
Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.

Show MeSH
Related in: MedlinePlus