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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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Gp39-mediated transcription antitermination by Tth RNAP at different intrinsic terminators. (A) The scheme of the terminator constructs shows positions of the transcription start site (+1), 20-nt-long U-less region, termination site (t) and the run-off (RO) transcript. The sequences of the terminators are shown below the scheme; the transcript release points are underlined. (B) Termination was assayed after addition of NTPs (10, 25, 50 or 250 µM) to stalled U21 TECs in the absence or in the presence of gp39. (C) Plot shows termination efficiencies at different NTP concentrations. The plot shows averages and standard deviations from three independent experiments.
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gkr1285-F1: Gp39-mediated transcription antitermination by Tth RNAP at different intrinsic terminators. (A) The scheme of the terminator constructs shows positions of the transcription start site (+1), 20-nt-long U-less region, termination site (t) and the run-off (RO) transcript. The sequences of the terminators are shown below the scheme; the transcript release points are underlined. (B) Termination was assayed after addition of NTPs (10, 25, 50 or 250 µM) to stalled U21 TECs in the absence or in the presence of gp39. (C) Plot shows termination efficiencies at different NTP concentrations. The plot shows averages and standard deviations from three independent experiments.

Mentions: Analysis of in vitro transcription by Thermus (Tth or Taq) RNAP in the presence of gp39 revealed, unexpectedly, that gp39 suppresses transcription termination (Figure 1; our unpublished data). To study this phenomenon, we analyzed Thermus RNAP transcription from four templates containing different intrinsic terminators with different hairpin stem lengths (ranging from 8 to 21 bp) and loop sizes (ranging from 3 to 10 nt) (Figure 1A). Two terminators tested, phage λ tR2 and tR′, are classic model terminators used to study E. coli RNAP termination; two others, t65 from Tth phage P23–45 and t6 from Tth phage ϕYS40 (23), were predicted based on sequence analysis. Each terminator was fused to the T7 A1 promoter, which allowed the preparation of stalled TECs containing 21-nt-long radiolabeled RNA, followed by addition of gp39 and unlabeled NTPs (see ‘Materials and Methods’ section for details).Figure 1.


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)

Gp39-mediated transcription antitermination by Tth RNAP at different intrinsic terminators. (A) The scheme of the terminator constructs shows positions of the transcription start site (+1), 20-nt-long U-less region, termination site (t) and the run-off (RO) transcript. The sequences of the terminators are shown below the scheme; the transcript release points are underlined. (B) Termination was assayed after addition of NTPs (10, 25, 50 or 250 µM) to stalled U21 TECs in the absence or in the presence of gp39. (C) Plot shows termination efficiencies at different NTP concentrations. The plot shows averages and standard deviations from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1285-F1: Gp39-mediated transcription antitermination by Tth RNAP at different intrinsic terminators. (A) The scheme of the terminator constructs shows positions of the transcription start site (+1), 20-nt-long U-less region, termination site (t) and the run-off (RO) transcript. The sequences of the terminators are shown below the scheme; the transcript release points are underlined. (B) Termination was assayed after addition of NTPs (10, 25, 50 or 250 µM) to stalled U21 TECs in the absence or in the presence of gp39. (C) Plot shows termination efficiencies at different NTP concentrations. The plot shows averages and standard deviations from three independent experiments.
Mentions: Analysis of in vitro transcription by Thermus (Tth or Taq) RNAP in the presence of gp39 revealed, unexpectedly, that gp39 suppresses transcription termination (Figure 1; our unpublished data). To study this phenomenon, we analyzed Thermus RNAP transcription from four templates containing different intrinsic terminators with different hairpin stem lengths (ranging from 8 to 21 bp) and loop sizes (ranging from 3 to 10 nt) (Figure 1A). Two terminators tested, phage λ tR2 and tR′, are classic model terminators used to study E. coli RNAP termination; two others, t65 from Tth phage P23–45 and t6 from Tth phage ϕYS40 (23), were predicted based on sequence analysis. Each terminator was fused to the T7 A1 promoter, which allowed the preparation of stalled TECs containing 21-nt-long radiolabeled RNA, followed by addition of gp39 and unlabeled NTPs (see ‘Materials and Methods’ section for details).Figure 1.

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