Characterization of plastid psbT sense and antisense RNAs

The plastid psbB operon is composed of the psbB, psbT, psbH, petB and petD genes. The psbN gene is located in the intergenic region between psbT and psbH on the opposite DNA strand. Transcription of psbN is under control of sigma factor 3 (SIG3) and psbN read-through transcription produces antisense RNA to psbT mRNA. To investigate on the question of whether psbT gene expression might be regulated by antisense RNA, we have characterized psbT sense and antisense RNAs. Mapping of 5' and 3'-ends by circular RT-PCR and /or 5'-RACE experiments reveal the existence of two different sense and antisense RNAs each, one limited to psbT RNA and a larger one that covers, in addition, part of the psbB coding region. Sense and antisense RNAs seem to form double-stranded RNA/RNA hybrids as indicated by nuclease digestion experiments followed by RT-PCR amplification to reveal nuclease resistant RNA. Western immunoblotting using antibodies made against PSBT protein and primer extension analysis of different plastid mRNA species and psbT antisense RNA suggest that sequestering of psbT mRNA by hybrid formation results in translational inactivation of the psbT mRNA and provides protection against nucleolytic degradation of mRNA during photooxydative stress conditions.