Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding

J Mol Biol. 1997 Oct 17;273(1):38-51. doi: 10.1006/jmbi.1997.1294.

Abstract

The hok/sok locus of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The locus specifies two RNAs, hok mRNA and Sok antisense RNA. The post-segregational killing mediated by hok/sok is governed by a complicated control mechanism that involves both post-transcriptional inhibition of translation by Sok-RNA and activation of hok translation by mRNA 3' processing. Sok-RNA inhibits translation of a reading frame (mok) that overlaps with hok, and translation of hok is coupled to translation of mok. In the inactive full-length hok mRNA, the translational activator element at the mRNA 5'-end (tac) is sequestered by the fold-back-inhibitory element located at the mRNA 3'-end (fbi). The 5' to 3' pairing locks the RNA in an inert configuration in which the SDmok and Sok-RNA target regions are sequestered. Here we show that the 3' processing leads to major structural rearrangements in the mRNA 5'-end. The structure of the refolded RNA explains activation of translation and antisense RNA binding. The refolded RNA contains an antisense RNA target stem-loop that presents the target nucleotides in a single-stranded conformation. The stem of the target hairpin contains SDmok and AUGmok in a paired configuration. Using toeprinting analysis, we show that this pairing keeps SDmok in an accessible configuration. Furthermore, a mutational analysis shows that an internal loop in the target stem is prerequisite for efficient translation and antisense RNA binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / genetics*
  • Bacterial Proteins / genetics*
  • Bacterial Toxins*
  • Base Sequence
  • Blotting, Northern
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli Proteins*
  • Gene Expression Regulation
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleic Acid Conformation*
  • Nucleic Acid Hybridization
  • Plasmids / genetics*
  • Protein Biosynthesis*
  • RNA
  • RNA, Antisense / genetics
  • RNA, Antisense / metabolism*
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Ribonuclease H / metabolism
  • Sequence Deletion
  • Transcription, Genetic

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • Escherichia coli Proteins
  • Hok protein, E coli
  • RNA, Antisense
  • RNA, Bacterial
  • RNA, Messenger
  • Sok antisense RNA, E coli
  • RNA
  • Ribonuclease H