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A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.

Berdygulova Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L - Nucleic Acids Res. (2012)

Bottom Line: Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center.However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity.To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Piscataway, NJ 08854, USA.

ABSTRACT
Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

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Effect of gp39 on σA-dependent pausing. (A) The scheme of the nucleic acid scaffold used for analysis of σA-dependent pausing. The −10-like pause-inducing sequence is boxed, RNA 3′-end positions in the starting 20-mer, paused and full-length transcripts are indicated below the scheme. (B) Analysis of σA-dependent pausing in the absence or in the presence of gp39. (C) The plot showing the pause efficiencies (the data from three independent experiments).
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gkr1285-F6: Effect of gp39 on σA-dependent pausing. (A) The scheme of the nucleic acid scaffold used for analysis of σA-dependent pausing. The −10-like pause-inducing sequence is boxed, RNA 3′-end positions in the starting 20-mer, paused and full-length transcripts are indicated below the scheme. (B) Analysis of σA-dependent pausing in the absence or in the presence of gp39. (C) The plot showing the pause efficiencies (the data from three independent experiments).

Mentions: The detailed conditions of the σ-dependent pausing assay in reconstituted TECs are described in (42). Briefly, the TECs were reconstituted from Tth core RNAP and DNA and RNA oligonucleotides shown on Figure 6A in the same way as in the case of the t65 terminator scaffold, and the σA-subunit was added to 1 µM. Reconstituted TECs were sorbed to Ni-NTA agarose, and transcription was performed at 37°C in the presence of 100 µM NTPs in transcription buffer containing 40 mM Tris–НСl pH 7.9, 40 mM KCl and 10 mM MgCl2. Gp39 was added to 10 µM 3 min before addition of NTPs.


A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.

Berdygulova Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L - Nucleic Acids Res. (2012)

Effect of gp39 on σA-dependent pausing. (A) The scheme of the nucleic acid scaffold used for analysis of σA-dependent pausing. The −10-like pause-inducing sequence is boxed, RNA 3′-end positions in the starting 20-mer, paused and full-length transcripts are indicated below the scheme. (B) Analysis of σA-dependent pausing in the absence or in the presence of gp39. (C) The plot showing the pause efficiencies (the data from three independent experiments).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351154&req=5

gkr1285-F6: Effect of gp39 on σA-dependent pausing. (A) The scheme of the nucleic acid scaffold used for analysis of σA-dependent pausing. The −10-like pause-inducing sequence is boxed, RNA 3′-end positions in the starting 20-mer, paused and full-length transcripts are indicated below the scheme. (B) Analysis of σA-dependent pausing in the absence or in the presence of gp39. (C) The plot showing the pause efficiencies (the data from three independent experiments).
Mentions: The detailed conditions of the σ-dependent pausing assay in reconstituted TECs are described in (42). Briefly, the TECs were reconstituted from Tth core RNAP and DNA and RNA oligonucleotides shown on Figure 6A in the same way as in the case of the t65 terminator scaffold, and the σA-subunit was added to 1 µM. Reconstituted TECs were sorbed to Ni-NTA agarose, and transcription was performed at 37°C in the presence of 100 µM NTPs in transcription buffer containing 40 mM Tris–НСl pH 7.9, 40 mM KCl and 10 mM MgCl2. Gp39 was added to 10 µM 3 min before addition of NTPs.

Bottom Line: Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center.However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity.To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Piscataway, NJ 08854, USA.

ABSTRACT
Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.

Show MeSH
Related in: MedlinePlus