Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.
Nucleic Acids Res
; 33(19): 6268-76, 2005.
Article
in En
| MEDLINE
| ID: mdl-16260475
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Transcription, Genetic
/
Open Reading Frames
/
Promoter Regions, Genetic
/
Escherichia coli
Type of study:
Diagnostic_studies
Language:
En
Journal:
Nucleic Acids Res
Year:
2005
Type:
Article
Affiliation country:
United States