Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III--the effect of dsRNA binding on gene expression.
RNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of lambdaN gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.
Dasgupta
S
S
Gene Regulation and Chromosome Biology Laboratory, ABL-Basic Research Program, NCI-FCRDC, Frederick, MD 21702, USA.
Fernandez
L
L
Kameyama
L
L
Inada
T
T
Nakamura
Y
Y
Pappas
A
A
Court
D L
DL
D000483:Alleles; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D009876:Operon; D010641:Phenotype; D010957:Plasmids; D017354:Point Mutation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D043244:Ribonuclease III; D001616:beta-Galactosidase / Q000378:metabolism
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D001616:beta-Galactosidase