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.
Case
C C
CC
Department of Microbiology, University of California, Los Angeles 90024.
Roels
S M
SM
Jensen
P D
PD
Lee
J
J
Kleckner
N
N
Simons
R W
RW
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements; D004722:Endoribonucleases; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005809:Genes, Regulator; D006207:Half-Life; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D015183:Restriction Mapping; D043244:Ribonuclease III
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D012313:RNA; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli