fhlA repression by OxyS RNA: Kissing complex formation at two sites results in a stable antisense-target RNA complex

Liron Argaman, Shoshy Altuvia*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

174 Scopus citations

Abstract

OxyS is a small untranslated RNA that is induced in response to oxidative stress in Escherichia coli. This small RNA acts as a global regulator affecting the expression of multiple genes. OxyS represses the translation of thlA, a transcriptional activator for formate metabolism. Previously, we have shown that fhlA repression by OxyS is mediated through base-pairing with a short sequence overlapping the ribosome binding site. Here we show that the OxyS-fhlA interaction involves a second site residing further downstream, within the coding region of fhlA. Mutations that disrupt pairing at this site affect the ability of OxyS to prevent 30 S ribosomes from binding to fhlA mRNA. Structure probing of fhlA mRNA demonstrates that both sites reside in the loops of two stem-loop structures. OxyS-fhlA pairing analysis shows that OxyS binds wild-type fhlA with an apparent dissociation constant of 25 nM, indicating that kissing complex formation between OxyS and fhlA results in a stable antisense-target complex. Mutations at either site, which disrupt pairing of OxyS to fhlA, decrease the stability of this complex. Our results indicate that kissing complex formation is sufficient to repress fhlA translation by OxyS. (C) 2000 Academic Press.

Original languageAmerican English
Pages (from-to)1101-1112
Number of pages12
JournalJournal of Molecular Biology
Volume300
Issue number5
DOIs
StatePublished - 28 Jul 2000

Bibliographical note

Funding Information:
We thank S. Ringquist and L. Gold for purified 30 S ribosome subunits. We also thank Gerhart Wagner for the advice with the binding assays, Hanah Margalit, Ester Yeger-Lotem and Maya Elgrably-Weiss for conducting the computer search of oxyS-target genes. This work was supported by Human Frontier Science Program, and by grant number 95–00092 from the United States-Israel Binational Science Foundation and by THE ISRAEL SCIENCE FOUNDATION founded by The Academy of Sciences and Humanities - Centers of Excellence Program (to S.A.).

Keywords

  • Antisense RNA
  • FhlA
  • Imperfect duplex
  • Kissing complex
  • OxyS

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