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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 targets the β flap domain of Thermus RNAP. (A) Analysis of the effects of gp39 on transcription termination by wild-type (WT) and Δβ flap Taq RNAPs at the λtR2 terminator. Transcription was performed at 25 µM NTPs. (B) Mapping of the gp39 interaction site in the β flap domain of Tth RNAP by a bacterial two-hybrid assay. The numbers at the right indicate the β-galactosidase activity measured in Miller units relative to the negative control and represent average values and standard deviations from at least three independent experiments. (C) The β flap domain structure [Tth TEC, 2O5I (21)].The positions of the analyzed deletions are indicated. The flap tip region (amino acids 762–786) is shown in light green.
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gkr1285-F3: Gp39 targets the β flap domain of Thermus RNAP. (A) Analysis of the effects of gp39 on transcription termination by wild-type (WT) and Δβ flap Taq RNAPs at the λtR2 terminator. Transcription was performed at 25 µM NTPs. (B) Mapping of the gp39 interaction site in the β flap domain of Tth RNAP by a bacterial two-hybrid assay. The numbers at the right indicate the β-galactosidase activity measured in Miller units relative to the negative control and represent average values and standard deviations from at least three independent experiments. (C) The β flap domain structure [Tth TEC, 2O5I (21)].The positions of the analyzed deletions are indicated. The flap tip region (amino acids 762–786) is shown in light green.

Mentions: To identify what surface of RNAP is contacted by gp39, we analyzed several Taq RNAP mutants harboring extensive deletions, (i) the β flap tip deletion (Δ757–793); (ii) the Thermus-specific β′ non-conserved domain (β′NCD) deletion (Δ159–451); and (iii) deletion of the αCTD (Δ231–315). Taq RNAP mutants with deletions of the β flap tip and αCTDs are unable to efficiently recognize −10/−35 promoters (24). Thus, to test termination by these RNAP mutants we used a DNA template that contained an extended −10 promoter, galP1, fused to the tR2 terminator. Whereas deletion of the β′NCD or αCTDs had no major effect on gp39 activity (Supplementary Figure S4), deletion of the β flap completely eliminated the ability of gp39 to antiterminate transcription (Figure 3A). Thus, we conclude that the β flap tip is essential for gp39-mediated antitermination.Figure 3.


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 targets the β flap domain of Thermus RNAP. (A) Analysis of the effects of gp39 on transcription termination by wild-type (WT) and Δβ flap Taq RNAPs at the λtR2 terminator. Transcription was performed at 25 µM NTPs. (B) Mapping of the gp39 interaction site in the β flap domain of Tth RNAP by a bacterial two-hybrid assay. The numbers at the right indicate the β-galactosidase activity measured in Miller units relative to the negative control and represent average values and standard deviations from at least three independent experiments. (C) The β flap domain structure [Tth TEC, 2O5I (21)].The positions of the analyzed deletions are indicated. The flap tip region (amino acids 762–786) is shown in light green.
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Related In: Results  -  Collection

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gkr1285-F3: Gp39 targets the β flap domain of Thermus RNAP. (A) Analysis of the effects of gp39 on transcription termination by wild-type (WT) and Δβ flap Taq RNAPs at the λtR2 terminator. Transcription was performed at 25 µM NTPs. (B) Mapping of the gp39 interaction site in the β flap domain of Tth RNAP by a bacterial two-hybrid assay. The numbers at the right indicate the β-galactosidase activity measured in Miller units relative to the negative control and represent average values and standard deviations from at least three independent experiments. (C) The β flap domain structure [Tth TEC, 2O5I (21)].The positions of the analyzed deletions are indicated. The flap tip region (amino acids 762–786) is shown in light green.
Mentions: To identify what surface of RNAP is contacted by gp39, we analyzed several Taq RNAP mutants harboring extensive deletions, (i) the β flap tip deletion (Δ757–793); (ii) the Thermus-specific β′ non-conserved domain (β′NCD) deletion (Δ159–451); and (iii) deletion of the αCTD (Δ231–315). Taq RNAP mutants with deletions of the β flap tip and αCTDs are unable to efficiently recognize −10/−35 promoters (24). Thus, to test termination by these RNAP mutants we used a DNA template that contained an extended −10 promoter, galP1, fused to the tR2 terminator. Whereas deletion of the β′NCD or αCTDs had no major effect on gp39 activity (Supplementary Figure S4), deletion of the β flap completely eliminated the ability of gp39 to antiterminate transcription (Figure 3A). Thus, we conclude that the β flap tip is essential for gp39-mediated antitermination.Figure 3.

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