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An Assay for Quantifying Protein-RNA Binding in Bacteria
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Published on: June 12, 2019

RNA-binding specificity of E. coli NusA.

Stefan Prasch1, Marcel Jurk, Robert S Washburn

  • 1Lehrstuhl für Struktur und Chemie der Biopolymere & Research Center for Bio-Macromolecules, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany. stefan.prasch@uni-bayreuth.de

Nucleic Acids Research
|June 12, 2009
PubMed
Summary

Bacteriophage lambda utilizes specific RNA sequences (nut regions) and E. coli proteins to form a termination-resistant complex. This study quantifies the binding of NusA protein to these RNA sequences, revealing key interactions for anti-termination.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • The nut regions (boxA, boxB, boxC) of phage lambda RNA are crucial for forming termination-resistant RNA polymerase complexes.
  • These complexes involve E. coli proteins (NusA, NusB, NusG, NusE) and the phage lambda N protein.
  • While NusB and NusE RNA binding is quantified, NusA's interaction with nut regions is less defined.

Purpose of the Study:

  • To quantitatively define the interaction between the E. coli NusA protein and the nut regions of phage lambda RNA.
  • To elucidate the role of the spacer sequence and specific boxA variants in NusA binding.
  • To understand how NusA binding affinities contribute to the assembly of the anti-termination complex.

Main Methods:

  • Isotropic and anisotropic fluorescence equilibrium titrations were employed to measure NusA-RNA binding.
  • Mutational analysis of the spacer sequence within the nut regions was performed.
  • Binding affinities (K(d) values) were determined for various RNA constructs, including nutR, nutL, and rrn boxA variants.

Main Results:

  • NusA specifically binds to the spacer sequence located between boxA and boxB in nutR and nutL regions.
  • Mutations within the spacer sequence abolish NusA complex formation.
  • NusA exhibits a 50% higher affinity for nutL compared to nutR.
  • rrn boxA binds NusA without a spacer, and with significantly lower K(d) values than nutR boxA-spacer.

Conclusions:

  • NusA binding is sequence-specific, primarily targeting the nut spacer, with variations in affinity between nutL and nutR.
  • Differences in NusA binding affinities, particularly with rrn boxA, may explain the necessity of lambda N protein for efficient anti-termination.
  • These findings provide quantitative insights into the assembly mechanism of the phage lambda anti-termination complex.