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RNA-binding specificity of E. coli NusA.

Prasch S, Jurk M, Washburn RS, Gottesman ME, Wöhrl BM, Rösch P - Nucleic Acids Res. (2009)

Bottom Line: The K(d) values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer.These differences may explain why lambda requires an additional protein, lambda N, to suppress termination.Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Struktur und Chemie der Biopolymere & Research Center for Bio-Macromolecules, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany. stefan.prasch@uni-bayreuth.de

ABSTRACT
The RNA sequences boxA, boxB and boxC constitute the nut regions of phage lambda. They nucleate the formation of a termination-resistant RNA polymerase complex on the lambda chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the lambda N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The K(d) values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why lambda requires an additional protein, lambda N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.

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Model of the anti-termination network. The interaction of the RNAP with various factors important for anti-termination (see text for details).
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Figure 6: Model of the anti-termination network. The interaction of the RNAP with various factors important for anti-termination (see text for details).

Mentions: Note that the NusE/NusB complex binds to boxA with affinities in the nanomolar range (5), whereas the Kd values for NusA–SKK are in the micromolar range. We suggest that tight RNA binding by NusA may not be required since it is already bound to RNAP and thus in close vicinity to nascent RNA. The λnutL spacer sequence differs from that of λnutR spacer (Table 1), and this difference is thought to account in part for the enhanced efficiency of Nun-mediated termination at λnutL relative to λnutR (Washburn, R.S. and Gottesman,M.E., unpublished data), and λnutL boxA-spacer binds with significantly higher affinity to NusA–SKK than does λnutR boxA-spacer. Both spacer sequences contain U's at residues 13 and 14, implying that these bases are important interaction partners. As shown above, replacement of the U's with G's completely abolished NusA–SKK binding to λnutL-spacer. From this and other data, the following picture of the assembly of the anti-termination complex at the λnut RNA has evolved (Figure 6): After RNAP has synthesized λnut RNA, NusE and NusB bind to boxA, and NusA binds to spacer facilitated by NusA AR2 interaction with the C-terminal domain of the α subunit of RNAP. λN protein binds to AR1 of NusA as demonstrated for N(34-47) (17,30), forming a weak helix at the protein's N-terminus (17). This weak helix facilitates recognition of boxB (17). NusA interaction with RNA is thus stabilized by the AR2:RNAP interaction as well as by the AR1:N:boxB interaction, relieving the requirement for tight binding of NusA to λnut.Figure 6.


RNA-binding specificity of E. coli NusA.

Prasch S, Jurk M, Washburn RS, Gottesman ME, Wöhrl BM, Rösch P - Nucleic Acids Res. (2009)

Model of the anti-termination network. The interaction of the RNAP with various factors important for anti-termination (see text for details).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724277&req=5

Figure 6: Model of the anti-termination network. The interaction of the RNAP with various factors important for anti-termination (see text for details).
Mentions: Note that the NusE/NusB complex binds to boxA with affinities in the nanomolar range (5), whereas the Kd values for NusA–SKK are in the micromolar range. We suggest that tight RNA binding by NusA may not be required since it is already bound to RNAP and thus in close vicinity to nascent RNA. The λnutL spacer sequence differs from that of λnutR spacer (Table 1), and this difference is thought to account in part for the enhanced efficiency of Nun-mediated termination at λnutL relative to λnutR (Washburn, R.S. and Gottesman,M.E., unpublished data), and λnutL boxA-spacer binds with significantly higher affinity to NusA–SKK than does λnutR boxA-spacer. Both spacer sequences contain U's at residues 13 and 14, implying that these bases are important interaction partners. As shown above, replacement of the U's with G's completely abolished NusA–SKK binding to λnutL-spacer. From this and other data, the following picture of the assembly of the anti-termination complex at the λnut RNA has evolved (Figure 6): After RNAP has synthesized λnut RNA, NusE and NusB bind to boxA, and NusA binds to spacer facilitated by NusA AR2 interaction with the C-terminal domain of the α subunit of RNAP. λN protein binds to AR1 of NusA as demonstrated for N(34-47) (17,30), forming a weak helix at the protein's N-terminus (17). This weak helix facilitates recognition of boxB (17). NusA interaction with RNA is thus stabilized by the AR2:RNAP interaction as well as by the AR1:N:boxB interaction, relieving the requirement for tight binding of NusA to λnut.Figure 6.

Bottom Line: The K(d) values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer.These differences may explain why lambda requires an additional protein, lambda N, to suppress termination.Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Struktur und Chemie der Biopolymere & Research Center for Bio-Macromolecules, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany. stefan.prasch@uni-bayreuth.de

ABSTRACT
The RNA sequences boxA, boxB and boxC constitute the nut regions of phage lambda. They nucleate the formation of a termination-resistant RNA polymerase complex on the lambda chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the lambda N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The K(d) values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why lambda requires an additional protein, lambda N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.

Show MeSH
Related in: MedlinePlus