HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli.
Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.
Koo
Jovanka T
JT
Department of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242, USA.
Choe
Juno
J
Moseley
Steve L
SL
D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism; D017101:Bacteriophage P1 / Q000235:genetics; D002874:Chromosome Mapping; D003227:Conjugation, Genetic; D053487:DEAD-box RNA Helicases; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009876:Operon; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014161:Transduction, Genetic
D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C080952:DaaE protein, E coli; D036781:Fimbriae Proteins; D004722:Endoribonucleases; C092662:hrpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases