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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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Influence of gp39 on transcription elongation by Tth RNAP. (A) Analysis of the average elongation rates of Tth RNAP on the λPR-rpoB template. Positions of the starting 26-mer and full-length run-off transcripts are indicated at the left; positions of several transcription pauses are indicated with asterisks. (B) Analysis of the rates of single nucleotide addition in complex of Tth RNAP with the minimal nucleic acid scaffold. (C) Effect of gp39 on the reaction of pyrophosphorolysis. Positions of the starting 10 nt RNA and 8/9 nt reaction products are shown on the left of the gel. The apparent kobs values (min−1) correspond to the rates of accumulation of the 8 nt RNA product. (D) Effect of gp39 on the reaction of intrinsic endonucleolytic RNA cleavage. The cleavage site of RNA in the synthetic scaffold is indicated by an arrow.
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gkr1285-F5: Influence of gp39 on transcription elongation by Tth RNAP. (A) Analysis of the average elongation rates of Tth RNAP on the λPR-rpoB template. Positions of the starting 26-mer and full-length run-off transcripts are indicated at the left; positions of several transcription pauses are indicated with asterisks. (B) Analysis of the rates of single nucleotide addition in complex of Tth RNAP with the minimal nucleic acid scaffold. (C) Effect of gp39 on the reaction of pyrophosphorolysis. Positions of the starting 10 nt RNA and 8/9 nt reaction products are shown on the left of the gel. The apparent kobs values (min−1) correspond to the rates of accumulation of the 8 nt RNA product. (D) Effect of gp39 on the reaction of intrinsic endonucleolytic RNA cleavage. The cleavage site of RNA in the synthetic scaffold is indicated by an arrow.

Mentions: The ability of gp39 to decrease RNAP pausing at the terminator suggested that it may also affect the rate of transcription elongation. To test this notion, we analyzed transcription on a DNA template containing a λ PR promoter fused to a 600-bp-long DNA fragment from the rpoB gene of E. coli. The rpoB template is devoid of strong pause signals and was previously used for comparison of transcription rates of various RNAPs (30,33). Gp39 increased the average elongation rate of Tth RNAP ∼2-fold: when transcription was performed at 55°C in the absence of gp39, most RNAPs reached the end of the template after ∼40 s, while in the presence of gp39 the reaction was complete by 20 s (Figure 5A). Similar 2-fold stimulatory effects of gp39 were observed when transcription was repeated at low temperatures at which the transcription rate of Thermus RNAP is significantly reduced (Supplementary Figure S8). Gp39 also significantly decreased the duration of transcription pauses (indicated with asterisks on Figure 5A) that were observed at several positions of this template. Thus, gp39 exhibits antipausing activity during both transcription elongation and termination and increases the average elongation rate.Figure 5.


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)

Influence of gp39 on transcription elongation by Tth RNAP. (A) Analysis of the average elongation rates of Tth RNAP on the λPR-rpoB template. Positions of the starting 26-mer and full-length run-off transcripts are indicated at the left; positions of several transcription pauses are indicated with asterisks. (B) Analysis of the rates of single nucleotide addition in complex of Tth RNAP with the minimal nucleic acid scaffold. (C) Effect of gp39 on the reaction of pyrophosphorolysis. Positions of the starting 10 nt RNA and 8/9 nt reaction products are shown on the left of the gel. The apparent kobs values (min−1) correspond to the rates of accumulation of the 8 nt RNA product. (D) Effect of gp39 on the reaction of intrinsic endonucleolytic RNA cleavage. The cleavage site of RNA in the synthetic scaffold is indicated by an arrow.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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gkr1285-F5: Influence of gp39 on transcription elongation by Tth RNAP. (A) Analysis of the average elongation rates of Tth RNAP on the λPR-rpoB template. Positions of the starting 26-mer and full-length run-off transcripts are indicated at the left; positions of several transcription pauses are indicated with asterisks. (B) Analysis of the rates of single nucleotide addition in complex of Tth RNAP with the minimal nucleic acid scaffold. (C) Effect of gp39 on the reaction of pyrophosphorolysis. Positions of the starting 10 nt RNA and 8/9 nt reaction products are shown on the left of the gel. The apparent kobs values (min−1) correspond to the rates of accumulation of the 8 nt RNA product. (D) Effect of gp39 on the reaction of intrinsic endonucleolytic RNA cleavage. The cleavage site of RNA in the synthetic scaffold is indicated by an arrow.
Mentions: The ability of gp39 to decrease RNAP pausing at the terminator suggested that it may also affect the rate of transcription elongation. To test this notion, we analyzed transcription on a DNA template containing a λ PR promoter fused to a 600-bp-long DNA fragment from the rpoB gene of E. coli. The rpoB template is devoid of strong pause signals and was previously used for comparison of transcription rates of various RNAPs (30,33). Gp39 increased the average elongation rate of Tth RNAP ∼2-fold: when transcription was performed at 55°C in the absence of gp39, most RNAPs reached the end of the template after ∼40 s, while in the presence of gp39 the reaction was complete by 20 s (Figure 5A). Similar 2-fold stimulatory effects of gp39 were observed when transcription was repeated at low temperatures at which the transcription rate of Thermus RNAP is significantly reduced (Supplementary Figure S8). Gp39 also significantly decreased the duration of transcription pauses (indicated with asterisks on Figure 5A) that were observed at several positions of this template. Thus, gp39 exhibits antipausing activity during both transcription elongation and termination and increases the average elongation rate.Figure 5.

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