Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

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Abstract
A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E.colitrp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased β-galactosidase synthesis, were selected for DNA sequencing. One clone has a G→A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C→T and T→C) between the Shine-Dalgarno sequence and initiation codon which raise expression of β-galactosidase two-fold. A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present. These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.