Limits...
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.

Show MeSH

Related in: MedlinePlus

Fluorescence anisotropy measurements with seperated RNAs regions. In each titration 50 nM of 6-FAM-labeled RNA was used. (A) 50 nM of 6-FAM-labeled λnutL boxA-spacer (squares), λnutL spacer (circles), λnutL boxA (triangles) were titrated with NusA-SKK. Kd values of 71 µM and 24 µM were determined for λnutL boxA-spacer, λnutL spacer, respectively (solid lines). No Kd value could be fitted to λnutL boxA (see Table 1). (B) 50 nM of 6-FAM-labeled λnutR boxA-spacer (circles), λnutR spacer (squares), λnutR boxA (triangles) were titrated with NusA-SKK. Kd values of 126 µM and 137 µM were determined for λnutR boxA-spacer, λnutR spacer, respectively (solid lines). No Kd value could be fitted to λnutR boxA (see Table 1). (C) 50 nM of 6-FAM-labeled rrn boxA alone (open triangle), rrn cac-boxA-spacer (open square), rrn spacer I (open circle), rrn spacer II (filled circle), rrn spacer III (filled triangle) were titrated with NusA-SKK. Kd values can be seen in Table 1. No Kd value could be fitted to rrn boxA (see Table 1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724277&req=5

Figure 3: Fluorescence anisotropy measurements with seperated RNAs regions. In each titration 50 nM of 6-FAM-labeled RNA was used. (A) 50 nM of 6-FAM-labeled λnutL boxA-spacer (squares), λnutL spacer (circles), λnutL boxA (triangles) were titrated with NusA-SKK. Kd values of 71 µM and 24 µM were determined for λnutL boxA-spacer, λnutL spacer, respectively (solid lines). No Kd value could be fitted to λnutL boxA (see Table 1). (B) 50 nM of 6-FAM-labeled λnutR boxA-spacer (circles), λnutR spacer (squares), λnutR boxA (triangles) were titrated with NusA-SKK. Kd values of 126 µM and 137 µM were determined for λnutR boxA-spacer, λnutR spacer, respectively (solid lines). No Kd value could be fitted to λnutR boxA (see Table 1). (C) 50 nM of 6-FAM-labeled rrn boxA alone (open triangle), rrn cac-boxA-spacer (open square), rrn spacer I (open circle), rrn spacer II (filled circle), rrn spacer III (filled triangle) were titrated with NusA-SKK. Kd values can be seen in Table 1. No Kd value could be fitted to rrn boxA (see Table 1).

Mentions: In the case of the λnut sites, we find that the λnutL spacer binds to NusA–SKK (24 µM), whereas boxA alone shows no association with the protein (Figure 3A). Similarly, the nutR spacer binds NusA-SKK with an affinity nearly identical to that of nutR boxA-spacer (Kd value ∼137 µM; Figure 3B), whereas NusA–SKK binding to boxA could not be detected.Figure 3.


RNA-binding specificity of E. coli NusA.

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

Fluorescence anisotropy measurements with seperated RNAs regions. In each titration 50 nM of 6-FAM-labeled RNA was used. (A) 50 nM of 6-FAM-labeled λnutL boxA-spacer (squares), λnutL spacer (circles), λnutL boxA (triangles) were titrated with NusA-SKK. Kd values of 71 µM and 24 µM were determined for λnutL boxA-spacer, λnutL spacer, respectively (solid lines). No Kd value could be fitted to λnutL boxA (see Table 1). (B) 50 nM of 6-FAM-labeled λnutR boxA-spacer (circles), λnutR spacer (squares), λnutR boxA (triangles) were titrated with NusA-SKK. Kd values of 126 µM and 137 µM were determined for λnutR boxA-spacer, λnutR spacer, respectively (solid lines). No Kd value could be fitted to λnutR boxA (see Table 1). (C) 50 nM of 6-FAM-labeled rrn boxA alone (open triangle), rrn cac-boxA-spacer (open square), rrn spacer I (open circle), rrn spacer II (filled circle), rrn spacer III (filled triangle) were titrated with NusA-SKK. Kd values can be seen in Table 1. No Kd value could be fitted to rrn boxA (see Table 1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Fluorescence anisotropy measurements with seperated RNAs regions. In each titration 50 nM of 6-FAM-labeled RNA was used. (A) 50 nM of 6-FAM-labeled λnutL boxA-spacer (squares), λnutL spacer (circles), λnutL boxA (triangles) were titrated with NusA-SKK. Kd values of 71 µM and 24 µM were determined for λnutL boxA-spacer, λnutL spacer, respectively (solid lines). No Kd value could be fitted to λnutL boxA (see Table 1). (B) 50 nM of 6-FAM-labeled λnutR boxA-spacer (circles), λnutR spacer (squares), λnutR boxA (triangles) were titrated with NusA-SKK. Kd values of 126 µM and 137 µM were determined for λnutR boxA-spacer, λnutR spacer, respectively (solid lines). No Kd value could be fitted to λnutR boxA (see Table 1). (C) 50 nM of 6-FAM-labeled rrn boxA alone (open triangle), rrn cac-boxA-spacer (open square), rrn spacer I (open circle), rrn spacer II (filled circle), rrn spacer III (filled triangle) were titrated with NusA-SKK. Kd values can be seen in Table 1. No Kd value could be fitted to rrn boxA (see Table 1).
Mentions: In the case of the λnut sites, we find that the λnutL spacer binds to NusA–SKK (24 µM), whereas boxA alone shows no association with the protein (Figure 3A). Similarly, the nutR spacer binds NusA-SKK with an affinity nearly identical to that of nutR boxA-spacer (Kd value ∼137 µM; Figure 3B), whereas NusA–SKK binding to boxA could not be detected.Figure 3.

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