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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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Analysis of the NusA effects on intrinsic termination and gp39-mediated antitermination. (A) Analysis of termination on the λtR2 (top) and t65 (bottom) terminators in the presence of gp39 (0.4 and 4 µM) and NusA (0.16 and 0.8 µM). Transcription was performed at 25 µM NTPs. (B) NusA effects on gp39-mediated antitermination at low NTP concentrations (2.5 µM). Gp39 and NusA were present at 1 µM and 0.16/0.8 µM. The plot shows averages from two independent experiments.
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gkr1285-F2: Analysis of the NusA effects on intrinsic termination and gp39-mediated antitermination. (A) Analysis of termination on the λtR2 (top) and t65 (bottom) terminators in the presence of gp39 (0.4 and 4 µM) and NusA (0.16 and 0.8 µM). Transcription was performed at 25 µM NTPs. (B) NusA effects on gp39-mediated antitermination at low NTP concentrations (2.5 µM). Gp39 and NusA were present at 1 µM and 0.16/0.8 µM. The plot shows averages from two independent experiments.

Mentions: The antitermination activities of λ phage antiterminators N and Q depend on NusA (see ‘Introduction’ section). We analyzed the effect of recombinant Tth NusA on transcript termination on tR2 and t65 in the presence or in the absence of gp39. In the presence of 25 µM NTPs and NusA, termination efficiencies on both terminators reached ∼100% (Figure 2A, lanes 4 and 5), compared to 25% for tR2 and 50% for t65 in the absence of NusA (Figure 2A, lane 1). The half termination-stimulatory concentration of NusA was <25 nM (Supplementary Figure S3A). Gp39 fully suppressed the effect of NusA and stimulated efficient terminator read-through (compare lanes 2, 3 and 6, 7 in Figure 2A). While the Q protein of lambdoid phage 82 (Q82) efficiently antiterminates at elevated NTP concentrations, it still requires NusA for antitermination at low NTP concentrations, when termination is more efficient (11). We therefore repeated the experiment with the tR2 terminator using low NTP concentrations (2.5 µM) (Figure 2B). Under these conditions, the efficiency of intrinsic termination reached 92%; NusA increased it to 98% while gp39 decreased the level of termination to ∼50% irrespective of the presence or the absence of NusA.Figure 2.


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)

Analysis of the NusA effects on intrinsic termination and gp39-mediated antitermination. (A) Analysis of termination on the λtR2 (top) and t65 (bottom) terminators in the presence of gp39 (0.4 and 4 µM) and NusA (0.16 and 0.8 µM). Transcription was performed at 25 µM NTPs. (B) NusA effects on gp39-mediated antitermination at low NTP concentrations (2.5 µM). Gp39 and NusA were present at 1 µM and 0.16/0.8 µM. The plot shows averages from two independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1285-F2: Analysis of the NusA effects on intrinsic termination and gp39-mediated antitermination. (A) Analysis of termination on the λtR2 (top) and t65 (bottom) terminators in the presence of gp39 (0.4 and 4 µM) and NusA (0.16 and 0.8 µM). Transcription was performed at 25 µM NTPs. (B) NusA effects on gp39-mediated antitermination at low NTP concentrations (2.5 µM). Gp39 and NusA were present at 1 µM and 0.16/0.8 µM. The plot shows averages from two independent experiments.
Mentions: The antitermination activities of λ phage antiterminators N and Q depend on NusA (see ‘Introduction’ section). We analyzed the effect of recombinant Tth NusA on transcript termination on tR2 and t65 in the presence or in the absence of gp39. In the presence of 25 µM NTPs and NusA, termination efficiencies on both terminators reached ∼100% (Figure 2A, lanes 4 and 5), compared to 25% for tR2 and 50% for t65 in the absence of NusA (Figure 2A, lane 1). The half termination-stimulatory concentration of NusA was <25 nM (Supplementary Figure S3A). Gp39 fully suppressed the effect of NusA and stimulated efficient terminator read-through (compare lanes 2, 3 and 6, 7 in Figure 2A). While the Q protein of lambdoid phage 82 (Q82) efficiently antiterminates at elevated NTP concentrations, it still requires NusA for antitermination at low NTP concentrations, when termination is more efficient (11). We therefore repeated the experiment with the tR2 terminator using low NTP concentrations (2.5 µM) (Figure 2B). Under these conditions, the efficiency of intrinsic termination reached 92%; NusA increased it to 98% while gp39 decreased the level of termination to ∼50% irrespective of the presence or the absence of NusA.Figure 2.

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