Analysis of the promoters and transcripts involved in IS10 anti-sense RNA control.

Genetic analysis of eleven mutations affecting the IS10 promoters, pIN and pOUT, involved in anti-sense RNA control of transposase gene expression, and characterization of the transcripts, reveal that: (i) The transposase message (RNA-IN) and the anti-sense RNA (RNA-OUT) have been unambiguously identified in vivo. (ii) Five mutations affect pIN activity, and establish that pIN is the only IS10 promoter transcribing the tnp gene, and the only such IS10 promoter that responds to DNA-adenine methylation. (iii) Six mutations alter pOUT activity, and establish that pOUT is the only IS10 promoter specifying the anti-sense RNA-OUT. (iv) The latter, however, need not be so: heterologous promoters, if properly positioned, can also specify active anti-sense RNAs. (v) These heterologously promoted anti-sense RNAs are processed to species closely resembling native RNA-OUT.

The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain.

IS10 transposition is regulated by an approximately 70 nt anti-sense RNA, RNA-OUT. RNA-OUT folds into a duplex 'stem-domain' topped by a loosely paired 'loop-domain'. The loop-domain is critical for RNA-RNA pairing per se; pairing initiates by interaction of the RNA-OUT loop with the 5' end of the target mRNA. We show here that RNA-OUT is unusually stable in vivo (half-life 60 min) and that this stability is conferred by specific features of the RNA-OUT stem-domain. One critical feature is stable base-pairing: mutations that disrupt stem pairing destabilize RNA-OUT in vivo and abolish anti-sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA-OUT resistant to 3' exoribonucleases. Other features of the stem-domain prevent this essential duplex from being an effective substrate for double-strand nucleases: two single base mutations disrupt antisense control by making RNA-OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA-OUT stability. Implications for IS10 biology and the design of efficient anti-sense RNAs are discussed.