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Nucleic Acids Research, 1989, Vol. 17, No. 8 2933-2945
© 1989

MOLECULAR BIOLOGY

In vivo gene expression directed by synthetic promoter constructions restricted to the –10 and –35 consensus hexamers of E.coli

Marie-Ange Jacquet*, Ricardo Ehrlich+ and Claude Reiss

Institut Jacques Monod, CNRS and Université Paris VII, Tour 43, 2 Place Jussieu 75251, Paris cédex 05, France

To whom correspondence should be addressed

Received February 2, 1989. Revised March 23, 1989. Accepted March 23, 1989.

Two synthetic DNA sequences, carrying no other known E.coli promoter element than the consensus hexamers (CH) TTGACA (CH–35) and TATAAT CH(–10), spaced by 17 bp, were inserted in pBR329, in a position enabling transcription of the complete Cmr gene. The region upstream of the Cmr transcription start was carefully cleared of w.t. promoter elements (full deletion of the wild type (w.t.) Cmr promoter upstream +2 and large portion of an upstream coding sequence). Both synthetic promoters, which differ only by the sequences of the spacers (non consensus, constrained in AT or GC) support in vivo high level Cmr gene expression. The GC rich spacer is associated with transcription start at the usual +1 position, but with the AT rich spacer, transcription starts at several places, mainly in CH(-10). Rearranged promoter sequences derived from the synthetic ones upon transformation with partly ligated plasimds, yield new insights on the role of the standard CH pair, the size of the spacer and the sequence downstream of CH(–10).

+On leave of absence: present address: Instituto de Investigaciones Biologicas Clemente Estable, Avda. Italia 3318, Montevideo and Facultad de Humanidades y Ciencas, Tristan, Narvaja 1674, Montevideo, Uruguay


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