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.
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.
Franch
T
T
Department of Molecular Biology, Odense University, Denmark.
Gultyaev
A P
AP
Gerdes
K
K
D017209:Apoptosis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins; D001483:Base Sequence; D015152:Blotting, Northern; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D012313:RNA; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C087025:Hok protein, E coli; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012333:RNA, Messenger; C087026:Sok antisense RNA, E coli; D012313:RNA; D016914:Ribonuclease H