Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III.
Members of the ribonuclease III family are the primary agents of double-stranded (ds) RNA processing in prokaryotic and eukaryotic cells. Bacterial RNase III orthologs cleave their substrates in a highly site-specific manner, which is necessary for optimal RNA function or proper decay rates. The processing reactivities of Escherichia coli RNase III substrates are determined in part by the sequence content of two discrete double-helical elements, termed the distal box (db) and proximal box (pb). A minimal substrate of E.coli RNase III, muR1.1 RNA, was characterized and used to define the db and pb sequence requirements for reactivity and their involvement in cleavage site selection. The reactivities of muR1.1 RNA sequence variants were examined in assays of cleavage and binding in vitro. The ability of all examined substitutions in the db to inhibit cleavage by weakening RNase III binding indicates that the db is a positive determinant of RNase III recognition, with the canonical UA/UG sequence conferring optimal recognition. A similar analysis showed that the pb also functions as a positive recognition determinant. It also was shown that the ability of the GC or CG bp substitution at a specific position in the pb to inhibit RNase III binding is due to the purine 2-amino group, which acts as a minor groove recognition antideterminant. In contrast, a GC or CG bp at the pb position adjacent to the scissile bond can suppress cleavage without inhibiting binding, and thus act as a catalytic antideterminant. It is shown that a single pb+db 'set' is sufficient to specify a cleavage site, supporting the primary function of the two boxes as positive recognition determinants. The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate.
Pertzev
Alexandre V
AV
Department of Chemistry, Temple University, Philadelphia, PA 19122, USA.
Nicholson
Allen W
AW
D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D029968:Escherichia coli Proteins / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D043244:Ribonuclease III / Q000378:metabolism*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D038621:Regulatory Sequences, Ribonucleic Acid; D043244:Ribonuclease III; C468366:ribonuclease III, E coli