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A highly conserved 6S RNA structure is required for regulation of transcription

Nature Structural & Molecular Biology volume 12, pages 313–319 (2005)Cite this article

Abstract

6S RNA, a highly abundant noncoding RNA, regulates transcription through interaction with RNA polymerase in Escherichia coli. Computer searches identified 6S RNAs widely among γ-proteobacteria. Biochemical approaches were required to identify more divergent 6S RNAs. Two Bacillus subtilis RNAs were found to interact with the housekeeping form of RNA polymerase, thereby establishing them as 6S RNAs. A third B. subtilis RNA was discovered with distinct RNA polymerase–binding activity. Phylogenetic comparison and analysis of mutant RNAs revealed that a conserved secondary structure containing a single-stranded central bulge within a highly double-stranded molecule was essential for 6S RNA function in vivo and in vitro. Reconstitution experiments established the marked specificity of 6S RNA interactions for σ70-RNA polymerase, as well as the ability of 6S RNA to directly inhibit transcription. These data highlight the critical importance of structural characteristics for 6S RNA activity.

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Figure 1: Analysis of total and immunoprecipitated RNAs.
Figure 2: Selected secondary structure predictions for 6S RNAs from diverse bacteria.
Figure 3: Analysis of mutant 6S RNA activity by coimmunoprecipitation of σ70 with core RNAP.
Figure 4: Examination of 6S RNA and mutants for inhibition of transcription in vivo and in vitro.
Figure 5: In vitro reconstitution to examine specificity of 6S RNA interactions with various forms of RNAP.
Figure 6: Analysis of 6S and mutants for Eσ70-binding activity.

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Acknowledgements

We thank G. Storz for supporting experiments conducted by K.M.W. at the US National Institutes of Health (NIH). We thank S. Stibitz for providing B. pertussis cells and the Bacillus Genetic Stock Center for B. subtilis strains; R. Burgess, D. Jin and C. Price for antibodies; and L. Anthony, R. Burgess and R. Landick for RNAP preparations. We thank R. Burgess, R. Gourse, R. Landick and G. Storz for helpful discussions. This work was supported by the NIH (GM67955).

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Authors and Affiliations

  1. Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, 53706, Wisconsin, USA

    Amy E Trotochaud & Karen M Wassarman

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  1. Amy E Trotochaud
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Correspondence to Karen M Wassarman.

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Supplementary information

Supplementary Fig. 1

Sequence alignment. (PDF 345 kb)

Supplementary Fig. 2

Secondary structure mapping. (PDF 1802 kb)

Supplementary Table 1

Oligonucleotide sequences. (PDF 33 kb)

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Trotochaud, A., Wassarman, K. A highly conserved 6S RNA structure is required for regulation of transcription. Nat Struct Mol Biol 12, 313–319 (2005). https://doi.org/10.1038/nsmb917

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