title: Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.
abstract: The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid. Eight clones were deliberately selected for high activity and 105 clones were selected at random. All eight of the high-activity clones carried promoters that were located upstream of an ORF. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 7 had inserts which also mapped upstream of an ORF, while 49 mapped within or downstream of ORFs. Surprisingly, the eight promoters selected for high activity matched the canonical sigma70 -35 and -10 sequences no better than sequences from the randomly-selected clones. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT-PCR, but defined transcripts were not detected by northern analysis. Our results indicate that the E.coli chromosome carries numerous -35 and -10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent.
authors.affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Building 18T, Room 101, Bethesda, MD 20892-5430, USA.
authors.forename: Mitsuoki
authors.initials: M
authors.lastname: Kawano
authors.forename: Gisela
authors.initials: G
authors.lastname: Storz
authors.forename: B Sridhar
authors.initials: BS
authors.lastname: Rao
authors.forename: Judah L
authors.initials: JL
authors.lastname: Rosner
authors.forename: Robert G
authors.initials: RG
authors.lastname: Martin
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012808:Sigma Factor / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D016372:RNA, Antisense; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases