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Context and conformation dictate function of a transcription antitermination switch

Nature Structural & Molecular Biology volume 10, pages 812–819 (2003)Cite this article

Abstract

In bacteriophage l, transcription elongation is regulated by the N protein, which binds a nascent mRNA hairpin (termed boxB) and enables RNA polymerase to read through distal terminators. We have examined the structure, energetics and in vivo function of a number of N–boxB complexes derived from in vitro protein selection. Trp18 fully stacks on the RNA loop in the wild-type structure, and can become partially or completely unstacked when the sequence context is changed three or four residues away, resulting in a recognition interface in which the best binding residues depend on the sequence context. Notably, in vivo antitermination activity correlates with the presence of a stacked aromatic residue at position 18, but not with N–boxB binding affinity. Our work demonstrates that RNA polymerase responds to subtle conformational changes in cis-acting regulatory complexes and that approximation of components is not sufficient to generate a fully functional transcription switch.

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Figure 1: Structure and function of the N protein.
Figure 2: Relative stability of N peptide–boxB RNA complexes that differ at positions 14, 15 and 18.
Figure 3: Structure and folding characteristics of N–boxB complexes as detected by NMR and fluorescence spectroscopy.
Figure 4: Antitermination activities of N mutants, as determined using E. coli strains carrying a two-plasmid reporter system22.
Figure 5: Energetic and structural correlation of antitermination activity.
Figure 6: Characteristics of N-boxB-NusA interactions.

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Acknowledgements

We thank N. Franklin for the gifts of the two-plasmid N expressor–β-galactosidase reporter constructs, plasmid pMS7 and N–tester strain, P. von Hippel for the gift of pET-N1 plasmid and N protein, P. Legault for sharing the coordinates of the λ phage N peptide–boxB RNA complex and J.H. Richards, C.S. Parker and members of the Roberts laboratory for helpful comments on the manuscript. This work was supported by grants from US National Science Foundation (NSF) and National Institutes of Health (NIH) to R.W.R. R.W.R. is an Alfred P. Sloan research fellow, A.F. is an ACS postdoctoral fellow and T.T.T. was supported by an NIH training grant.

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  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Tianbing Xia, Adam Frankel, Terry T Takahashi & Richard W Roberts

  2. Changchun Institute of Applied Chemistry, Changchun, 130022, Jilin, China

    Jinsong Ren

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Xia, T., Frankel, A., Takahashi, T. et al. Context and conformation dictate function of a transcription antitermination switch. Nat Struct Mol Biol 10, 812–819 (2003). https://doi.org/10.1038/nsb983

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