Open AccessBiological expression of an Escherichia coli consensus sequence promoter and some mutant derivatives.
Author(s) -
John J. Rossi,
Xavier Soberón,
Yasumasa Marumoto,
jeannette mcmahon,
Keiichi Itakura
Publication year - 1983
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.80.11.3203
Subject(s) - transversion , biology , mutant , consensus sequence , microbiology and biotechnology , promoter , rna polymerase , base pair , mutagenesis , transcription (linguistics) , nucleic acid sequence , gene , plasmid , site directed mutagenesis , escherichia coli , genetics , mutation , gene expression , peptide sequence , linguistics , philosophy
A prokaryotic consensus sequence promoter has been chemically synthesized and cloned in bacterial plasmid vectors. This designed sequence is biologically active and promotes efficient expression of the genes to which it is fused. It is an unusually strong promoter in vitro, capable of specifying multiple rounds of transcription even when there is a large molar excess of heparin present prior to the addition of RNA polymerase. These properties make this a useful sequence for the in vitro production of RNAs. A 2-base-pair spacer mutant and a -35 region transversion mutant have been created in vitro in the synthetic promoter by synthetic-DNA-mediated, site-specific mutagenesis. The spacer mutant has a marginal in vivo effect on promoter strength but virtually abolishes the in vitro heparin resistance. The -35 region transversion changes a highly conserved nucleotide into the statistically least preferred base. This mutation has no marked effect on in vivo or in vitro promoter strength.