Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs-commonalities and differences

Olga Y. Burenina, Philipp G. Hoch, Katrin Damm, Margarita Salas, Timofei S. Zatsepin, Marcus Lechner, Tatiana S. Oretskaya, Elena A. Kubareva, Roland K. Hartmann

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

Bacterial 6S RNAs bind to the housekeeping RNA polymerase (σ;A-RNAP in Bacillus subtilis) to regulate transcription in a growth phase-dependent manner. B. subtilis expresses two 6S RNAs, 6S-1 and 6S-2 RNA, with different expression profiles. We show in vitro that 6S-2 RNA shares hallmark features with 6S-1 RNA: Both (1) are able to serve as templates for pRNA transcription; (2) bind with comparable affinity to σ;A-RNAP; (3) are able to specifically inhibit transcription from DNA promoters, and (4) can form stable 6S RNA:pRNA hybrid structures that (5) abolish binding to σ;A-RNAP. However, pRNAs of equal length dissociate faster from 6S-2 than 6S-1 RNA, owing to the higher A,U-content of 6S-2 pRNAs. This could have two mechanistic implications: (1) Short 6S-2 pRNAs (<10 nt) dissociate faster instead of being elongated to longer pRNAs, which could make it more difficult for 6S-2 RNA-stalled RNAP molecules to escape from the sequestration; and (2) relative to 6S-1 RNA, 6S-2 pRNAs of equal length will dissociate more rapidly from 6S-2 RNA after RNAP release, which could affect pRNA turnover or the kinetics of 6S-2 RNA binding to a new RNAP molecule. As 6S-2 pRNAs have not yet been detected in vivo, we considered that cellular RNAP release from 6S-2 RNA might occur via 6S-1 RNA displacing 6S-2 RNA from the enzyme, either in the absence of pRNA transcription or upon synthesis of very short 6S-2 pRNAs (5-mers, which would escape detection by deep sequencing). However, binding competition experiments argued against these possibilities.

Original languageEnglish
Pages (from-to)348-359
Number of pages12
JournalRNA
Volume20
Issue number3
DOIs
Publication statusPublished - Mar 2014
Externally publishedYes

Keywords

  • 6S RNA:pRNA hybrid stability
  • 6S-1 RNA
  • 6S-2 RNA
  • 6S-2 RNA release from RNAP
  • Affinity for σ;A-RNAP
  • BsrA
  • BsrB
  • PRNA transcripts

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