DNA sequences in gal operon override transcription elongation blocks.
The DNA loop that represses transcription from galactose (gal) promoters is infrequently formed in stationary-phase cells because the concentration of the loop architectural protein HU is significantly low at that state, resulting in expression of the operon in the absence of the gal inducer D-galactose. Unexpectedly, transcription from the gal promoters under these conditions overrides physical block because of the presence of the Gal repressor bound to an internal operator (O(I)) located downstream of the promoters. We have shown here that although a stretch of pyrimidine residues (UUCU) in the RNA:DNA hybrid located immediately upstream of O(I) weakens the RNA:DNA hybrid and favors RNA polymerase (RNAP) pausing and backtracking, a stretch of purines (GAGAG) in the RNA present immediately upstream of the pause sequence in the hybrid acts as an antipause element by stabilizing the RNA:DNA duplex and preventing backtracking. This facilitates forward translocation of RNAP, including overriding of the DNA-bound Gal repressor barrier at O(I). When the GAGAG sequence is separated from the pyrimidine sequence by a 5-bp DNA insertion, RNAP backtracking is favored from a weak hybrid to a more stable hybrid. RNAP backtracking is sensitive to Gre factors, D-galactose, and antisense oligonucleotides. The ability of a native DNA sequence to override transcription elongation blocks in the gal operon uncovers a previously unknown way of regulating gal metabolism in Escherichia coli. It also explains the synthesis of gal enzymes in the absence of inducer for biosynthetic reactions.
Lewis
Dale E A
DE
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA. aed@helix.nih.gov
Komissarova
Natalia
N
Le
Phuoc
P
Kashlev
Mikhail
M
Adhya
Sankar
S
D024509:3' Flanking Region / Q000235:genetics; D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D009876:Operon; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; D016376:Oligonucleotides, Antisense; D012329:RNA, Bacterial; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D005690:Galactose