Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.
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.
Kawano
Mitsuoki
M
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Building 18T, Room 101, Bethesda, MD 20892-5430, USA.
Storz
Gisela
G
Rao
B Sridhar
BS
Rosner
Judah L
JL
Martin
Robert G
RG
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
D016372:RNA, Antisense; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases