Biological expression of an Escherichia coli consensus sequence promoter and some mutant derivatives.
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.
D001483:Base Sequence; D003001:Cloning, Molecular; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D009876:Operon; D010957:Plasmids; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes