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N protein from lambdoid phages transforms NusA into an antiterminator by modulating NusA-RNA polymerase flap domain interactions.

Mishra S, Sen R - Nucleic Acids Res. (2015)

Bottom Line: Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination.The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain.We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Tuljaguda Complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad 500 001, India Graduate Studies, Manipal University, India.

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Involvement of β-flap domain in N antitermination. (A) Structural model of the EC developed through a hybrid approach combining X-ray crystallography, ab initio structural prediction, homology modeling and single-particle cryo-electron microscopy (29). Relevant portions of the structure have been highlighted. The flap domain is shown in green. DNA is shown as red double helix, whereas the nascent RNA (up to -16th residue) is in blue. The RNA is shown to be coming out of the exit channel. Different RNAP mutants are indicated in colored spheres. P251L and P254L are in β′-subunit. Others are in β-subunit. Active site Mg(II) is in violet sphere. Rest of the RNAP is shown as light gray in the background. An insert in the flap domain is shown in sky blue. (B) Autoradiogram showing the transcription read-through activities of the WT and the flap mutants at the triple terminators, TR′, T1 and T2. Concentrations of N in each lane are indicated. The schematic of the DNA template is shown next to the gel. H-19B nutR site is cloned upstream of the terminator cassette. Transcription was initiated from T7A1 promoter. NusG was added to the reactions to stimulate the H-19B N in vitro activity (21). RO denotes the amounts of transcript reached at the end of the template. Amounts of read-through (%RT) products at the end of the triple terminators (C) and after the TR′ terminator (D) are plotted against increasing concentrations of N. %RT was calculated as for (C), [RO]/[TR′ + T1 + T2 + RO] and for (D), [T1 + T2 + RO]/[TR′ + T1 + T2 + RO]. (E) Bar diagram showing the β−galactosidase activities from the reporter strain expressing indicated combinations of different RNAP and NusA derivatives. LacZYA reporter is cloned downstream of the H-19B nutR-TR′ sequence. Activities were measured both in the presence and the absence of N protein. Errors were calculated from the activities obtained from five to six independent colonies.
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Figure 2: Involvement of β-flap domain in N antitermination. (A) Structural model of the EC developed through a hybrid approach combining X-ray crystallography, ab initio structural prediction, homology modeling and single-particle cryo-electron microscopy (29). Relevant portions of the structure have been highlighted. The flap domain is shown in green. DNA is shown as red double helix, whereas the nascent RNA (up to -16th residue) is in blue. The RNA is shown to be coming out of the exit channel. Different RNAP mutants are indicated in colored spheres. P251L and P254L are in β′-subunit. Others are in β-subunit. Active site Mg(II) is in violet sphere. Rest of the RNAP is shown as light gray in the background. An insert in the flap domain is shown in sky blue. (B) Autoradiogram showing the transcription read-through activities of the WT and the flap mutants at the triple terminators, TR′, T1 and T2. Concentrations of N in each lane are indicated. The schematic of the DNA template is shown next to the gel. H-19B nutR site is cloned upstream of the terminator cassette. Transcription was initiated from T7A1 promoter. NusG was added to the reactions to stimulate the H-19B N in vitro activity (21). RO denotes the amounts of transcript reached at the end of the template. Amounts of read-through (%RT) products at the end of the triple terminators (C) and after the TR′ terminator (D) are plotted against increasing concentrations of N. %RT was calculated as for (C), [RO]/[TR′ + T1 + T2 + RO] and for (D), [T1 + T2 + RO]/[TR′ + T1 + T2 + RO]. (E) Bar diagram showing the β−galactosidase activities from the reporter strain expressing indicated combinations of different RNAP and NusA derivatives. LacZYA reporter is cloned downstream of the H-19B nutR-TR′ sequence. Activities were measured both in the presence and the absence of N protein. Errors were calculated from the activities obtained from five to six independent colonies.

Mentions: Earlier, we have isolated a RNAP β-subunit flap domain mutant, G1045D, together with β′-subunit mutants located in and around the RNA exit channel (P251S, P254L and R270C) that were defective for N-mediated antitermination (21). In the same study, a suppressor, L108F, in the CTD (the RNAP-binding domain) of H-19B N was also found to specifically suppress the defects of the RNAP β′-subunit mutants. Interestingly, it was unable to suppress the antitermination defect of G1045D mutant. Also in the model structure of the EC of the E. coli RNAP (Figure 2A), G1045D bearing part of the flap-domain is located away from the exit channel, which is the proposed area through where N approaches the interior of the EC (25). Therefore, G1045D mutation most likely does not directly affect the binding of C-terminal domain of the N protein to the RNAP.


N protein from lambdoid phages transforms NusA into an antiterminator by modulating NusA-RNA polymerase flap domain interactions.

Mishra S, Sen R - Nucleic Acids Res. (2015)

Involvement of β-flap domain in N antitermination. (A) Structural model of the EC developed through a hybrid approach combining X-ray crystallography, ab initio structural prediction, homology modeling and single-particle cryo-electron microscopy (29). Relevant portions of the structure have been highlighted. The flap domain is shown in green. DNA is shown as red double helix, whereas the nascent RNA (up to -16th residue) is in blue. The RNA is shown to be coming out of the exit channel. Different RNAP mutants are indicated in colored spheres. P251L and P254L are in β′-subunit. Others are in β-subunit. Active site Mg(II) is in violet sphere. Rest of the RNAP is shown as light gray in the background. An insert in the flap domain is shown in sky blue. (B) Autoradiogram showing the transcription read-through activities of the WT and the flap mutants at the triple terminators, TR′, T1 and T2. Concentrations of N in each lane are indicated. The schematic of the DNA template is shown next to the gel. H-19B nutR site is cloned upstream of the terminator cassette. Transcription was initiated from T7A1 promoter. NusG was added to the reactions to stimulate the H-19B N in vitro activity (21). RO denotes the amounts of transcript reached at the end of the template. Amounts of read-through (%RT) products at the end of the triple terminators (C) and after the TR′ terminator (D) are plotted against increasing concentrations of N. %RT was calculated as for (C), [RO]/[TR′ + T1 + T2 + RO] and for (D), [T1 + T2 + RO]/[TR′ + T1 + T2 + RO]. (E) Bar diagram showing the β−galactosidase activities from the reporter strain expressing indicated combinations of different RNAP and NusA derivatives. LacZYA reporter is cloned downstream of the H-19B nutR-TR′ sequence. Activities were measured both in the presence and the absence of N protein. Errors were calculated from the activities obtained from five to six independent colonies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Involvement of β-flap domain in N antitermination. (A) Structural model of the EC developed through a hybrid approach combining X-ray crystallography, ab initio structural prediction, homology modeling and single-particle cryo-electron microscopy (29). Relevant portions of the structure have been highlighted. The flap domain is shown in green. DNA is shown as red double helix, whereas the nascent RNA (up to -16th residue) is in blue. The RNA is shown to be coming out of the exit channel. Different RNAP mutants are indicated in colored spheres. P251L and P254L are in β′-subunit. Others are in β-subunit. Active site Mg(II) is in violet sphere. Rest of the RNAP is shown as light gray in the background. An insert in the flap domain is shown in sky blue. (B) Autoradiogram showing the transcription read-through activities of the WT and the flap mutants at the triple terminators, TR′, T1 and T2. Concentrations of N in each lane are indicated. The schematic of the DNA template is shown next to the gel. H-19B nutR site is cloned upstream of the terminator cassette. Transcription was initiated from T7A1 promoter. NusG was added to the reactions to stimulate the H-19B N in vitro activity (21). RO denotes the amounts of transcript reached at the end of the template. Amounts of read-through (%RT) products at the end of the triple terminators (C) and after the TR′ terminator (D) are plotted against increasing concentrations of N. %RT was calculated as for (C), [RO]/[TR′ + T1 + T2 + RO] and for (D), [T1 + T2 + RO]/[TR′ + T1 + T2 + RO]. (E) Bar diagram showing the β−galactosidase activities from the reporter strain expressing indicated combinations of different RNAP and NusA derivatives. LacZYA reporter is cloned downstream of the H-19B nutR-TR′ sequence. Activities were measured both in the presence and the absence of N protein. Errors were calculated from the activities obtained from five to six independent colonies.
Mentions: Earlier, we have isolated a RNAP β-subunit flap domain mutant, G1045D, together with β′-subunit mutants located in and around the RNA exit channel (P251S, P254L and R270C) that were defective for N-mediated antitermination (21). In the same study, a suppressor, L108F, in the CTD (the RNAP-binding domain) of H-19B N was also found to specifically suppress the defects of the RNAP β′-subunit mutants. Interestingly, it was unable to suppress the antitermination defect of G1045D mutant. Also in the model structure of the EC of the E. coli RNAP (Figure 2A), G1045D bearing part of the flap-domain is located away from the exit channel, which is the proposed area through where N approaches the interior of the EC (25). Therefore, G1045D mutation most likely does not directly affect the binding of C-terminal domain of the N protein to the RNAP.

Bottom Line: Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination.The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain.We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Tuljaguda Complex, 4-1-714 Mozamjahi Road, Nampally, Hyderabad 500 001, India Graduate Studies, Manipal University, India.

Show MeSH
Related in: MedlinePlus