Nucleic Acids Research, 1990, Vol. 18, No. 24 7367-7372
© 1990
Articles |
Promoter selectivity of Escherichia coli RNA polymerase: effect of base substitutions in the promoter –35 region on promoter strength+
Department of Molecular Genetics, National Institute of Genetics Mishima, Shizuoka 411, Japan
*To whom correspondence should be addressed
Received August 20, 1990. Revised October 30, 1990. Accepted October 30, 1990.
A set of 18 variant lac UV5 promoters was constructed, each carrying a single base substitution within the –35 region (nucleotide positions from –36 to –31 relative to the transcription start site). Using truncated DNA fragments carrying these variant promoters and purified Escherichia coil RNA polymerase holoenzyme, in vitro mixed transcription assays were performed to determine two parameters governing promoter strength: i.e., the binding affinity to RNA polymerase (parameter I) and the rate of open complex formation (parameter II). The following conclusions were drawn from the data presented: (1) Alteration in the promoter strength of variant promoters is dependent on both the position and base species of substitutions; (2) the consensus sequence (TTGACA) exhibits the highest values for both parameters; (3) base substitutions at nucleotide position –34 cause marked effect on both parameters; (4) cytosine at nucleotide position –32 can not be replaced with other nucleotides without significant reduction of the promoter strength; and (5) base substitution at nucleotide position –31 exerts only a little effect on parameter I. All these findings were confirmed by abortive initiation assays.
+ This is Paper 8 in the series entitled ‘Promoter Selectivity of Escherichia coli RNA Po1ymerase’. Paper 7 is Ref. 16.
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