Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates.
The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence. Instead, the [4/5] asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop. The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex. The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry. The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.
Calin-Jageman
Irina
I
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202, USA.
Nicholson
Allen W
AW
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005095:Exoribonucleases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D005095:Exoribonucleases; C022535:exoribonuclease II