Second-site suppression of RNase E essentiality by mutation of the deaD RNA helicase in Escherichia coli.
Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.
Department of Genetics, School of Medicine, Stanford University, Stanford, California, USA.
Masaru
M
Tamura
Johan A
JA
Kers
Stanley N
SN
Cohen
D002876:Chromosomes, Bacterial; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics
D029968:Escherichia coli Proteins; D004722:Endoribonucleases; C081905:rng protein, E coli; C022537:ribonuclease E; C088840:deaD protein, E coli; D053487:DEAD-box RNA Helicases