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A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination.

Muteeb G, Dey D, Mishra S, Sen R - Nucleic Acids Res. (2012)

Bottom Line: This interaction becomes essential when the elongation complex moves away from the nutR site.From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway.We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

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.

ABSTRACT
One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

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Effect of N on the rate of ATP hydrolysis by Rho. Amounts of [γ-32P]ATP hydrolysed by Rho in nanomoles are plotted against time both in the absence and presence of WT H-19B N. These assays were performed on the nascent RNA coming out of the stalled ECs formed (A) on single terminator (as in Figure 3A) and (B) on double terminator (as in Figure 3B) templates.100 nM each of Rho and H-19B N were used. In all, 300 nM NusA and 200 nM NusG were also present in these assays. The initial rates of ATP hydrolysis are indicated by dashed lines. Same experiments performed on ECs stalled at the single terminator (C) or double terminator template (D) (as in Figure 3) using P235H Rho. Rates of ATP hydrolysis by WT Rho are indicated by solid/dotted lines in (C) and (D). The rate values are stated in the panels.
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gks872-F4: Effect of N on the rate of ATP hydrolysis by Rho. Amounts of [γ-32P]ATP hydrolysed by Rho in nanomoles are plotted against time both in the absence and presence of WT H-19B N. These assays were performed on the nascent RNA coming out of the stalled ECs formed (A) on single terminator (as in Figure 3A) and (B) on double terminator (as in Figure 3B) templates.100 nM each of Rho and H-19B N were used. In all, 300 nM NusA and 200 nM NusG were also present in these assays. The initial rates of ATP hydrolysis are indicated by dashed lines. Same experiments performed on ECs stalled at the single terminator (C) or double terminator template (D) (as in Figure 3) using P235H Rho. Rates of ATP hydrolysis by WT Rho are indicated by solid/dotted lines in (C) and (D). The rate values are stated in the panels.

Mentions: ATPase activity of the WT Rho protein was measured from the release of inorganic phosphate (Pi) from ATP after separating on the polyethyleneimine (PEI)-cellulose TLC plates (Merck) with 0.75 M KH2PO4 (pH 3.5) as the mobile phase. In all the assays, the composition of the reaction mixture was 25 mM Tris–HCl (pH 8.0), 50 mM KCl and 5 mM MgCl2, 1 mM DTT and 0.1 mg/ml of BSA. Assays were performed on the nascent RNA emerging out of the transcription EC. Stalled ECs were formed at the lac operator site on the T7A1-nutR/tR1-lacO or T7A1-nutR/tR1-trpt'-lacO templates in the same way as described earlier. These complexes were incubated with 100 nM Rho in presence of 1 mM NTPs and [γ-32P]ATP (3000 Ci/mmol). Aliquots were removed and mixed with 1.5 M formic acid at various time points to stop the reaction. Release of Pi was analysed by exposing the TLC sheets to a Phosphorimager screen for 5 min and subsequently by scanning using Typhoon 9200 (Amersham), and the intensities of ATP and Pi were quantified by Image QuantTL software (Figure 4; Supplementary Figure S2 and S3).Figure 4.


A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination.

Muteeb G, Dey D, Mishra S, Sen R - Nucleic Acids Res. (2012)

Effect of N on the rate of ATP hydrolysis by Rho. Amounts of [γ-32P]ATP hydrolysed by Rho in nanomoles are plotted against time both in the absence and presence of WT H-19B N. These assays were performed on the nascent RNA coming out of the stalled ECs formed (A) on single terminator (as in Figure 3A) and (B) on double terminator (as in Figure 3B) templates.100 nM each of Rho and H-19B N were used. In all, 300 nM NusA and 200 nM NusG were also present in these assays. The initial rates of ATP hydrolysis are indicated by dashed lines. Same experiments performed on ECs stalled at the single terminator (C) or double terminator template (D) (as in Figure 3) using P235H Rho. Rates of ATP hydrolysis by WT Rho are indicated by solid/dotted lines in (C) and (D). The rate values are stated in the panels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526286&req=5

gks872-F4: Effect of N on the rate of ATP hydrolysis by Rho. Amounts of [γ-32P]ATP hydrolysed by Rho in nanomoles are plotted against time both in the absence and presence of WT H-19B N. These assays were performed on the nascent RNA coming out of the stalled ECs formed (A) on single terminator (as in Figure 3A) and (B) on double terminator (as in Figure 3B) templates.100 nM each of Rho and H-19B N were used. In all, 300 nM NusA and 200 nM NusG were also present in these assays. The initial rates of ATP hydrolysis are indicated by dashed lines. Same experiments performed on ECs stalled at the single terminator (C) or double terminator template (D) (as in Figure 3) using P235H Rho. Rates of ATP hydrolysis by WT Rho are indicated by solid/dotted lines in (C) and (D). The rate values are stated in the panels.
Mentions: ATPase activity of the WT Rho protein was measured from the release of inorganic phosphate (Pi) from ATP after separating on the polyethyleneimine (PEI)-cellulose TLC plates (Merck) with 0.75 M KH2PO4 (pH 3.5) as the mobile phase. In all the assays, the composition of the reaction mixture was 25 mM Tris–HCl (pH 8.0), 50 mM KCl and 5 mM MgCl2, 1 mM DTT and 0.1 mg/ml of BSA. Assays were performed on the nascent RNA emerging out of the transcription EC. Stalled ECs were formed at the lac operator site on the T7A1-nutR/tR1-lacO or T7A1-nutR/tR1-trpt'-lacO templates in the same way as described earlier. These complexes were incubated with 100 nM Rho in presence of 1 mM NTPs and [γ-32P]ATP (3000 Ci/mmol). Aliquots were removed and mixed with 1.5 M formic acid at various time points to stop the reaction. Release of Pi was analysed by exposing the TLC sheets to a Phosphorimager screen for 5 min and subsequently by scanning using Typhoon 9200 (Amersham), and the intensities of ATP and Pi were quantified by Image QuantTL software (Figure 4; Supplementary Figure S2 and S3).Figure 4.

Bottom Line: This interaction becomes essential when the elongation complex moves away from the nutR site.From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway.We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

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.

ABSTRACT
One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

Show MeSH
Related in: MedlinePlus