A mutant spacer sequence between -35 and -10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent.
To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ((-20)CCGGCTCG(-13)) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a -35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.
Liu
Mofang
M
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Tolstorukov
Michael
M
Zhurkin
Victor
V
Garges
Susan
S
Adhya
Sankar
S
D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007763:Lac Operon / Q000235:genetics*; D009154:Mutation; D011196:Potassium Permanganate / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D011953:Receptors, Cyclic AMP; D011196:Potassium Permanganate; D004247:DNA; D012321:DNA-Directed RNA Polymerases