Translational control of mRNA processing in the F1845 fimbrial operon of Escherichia coli.
Endoribonucleolytic processing followed by differential decay of the cleavage products is an increasingly recognized mechanism for achieving co-ordinate regulation of functionally related proteins encoded by bacterial polycistronic transcripts. Unlike most examples when RNases E or III initiate decay, the daa transcript encoding F1845 fimbriae, a member of the Dr family of adhesins in Escherichia coli, is processed by an as yet unidentified endoribonuclease using a unique recognition mechanism. An open reading frame (ORF) predicted to encode a 57-amino-acid polypeptide was identified flanking the daa processing site. To determine whether this ORF is involved in processing, site-directed mutagenesis was used to generate mutants with altered translational efficiencies. A mutation in the putative ribosome binding site preceding the ORF significantly inhibited processing while the introduction of a premature stop codon abolished processing. Site-directed mutagenesis was used to introduce a limited number of mutations into the ORF, designated daaP, to alter the reading frame such that a different polypeptide of a similar size was encoded. Despite the presumed presence of trafficking ribosomes, this mutant failed to be processed, suggesting that the sequence of the DaaP peptide is important. However, the failure of a wild-type copy of the daaP gene to complement these mutations in trans suggested that the presence of wild-type daaP gene product was not sufficient to promote processing. Although active translation has been found to inhibit processing by RNases E and III, our data suggest that translation of the daaP gene is required in cis to promote processing by the endonuclease, perhaps due to an interaction of the nascent peptide with the ribosome or the daaP mRNA.
Loomis
W P
WP
Department of Microbiology, University of Washington, Seattle 98195-7242, USA.
Moseley
S L
SL
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D010861:Fimbriae, Bacterial / Q000235:genetics*; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D009876:Operon; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial; D012333:RNA, Messenger
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger