Saturation mutagenesis of the octopine synthase enhancer: correlation of mutant phenotypes with binding of a nuclear protein factor

Abstract

A 16-base-pair palindrome from the Agrobacterium tumefaciens octopine synthase gene functions as a constitutive enhancer in plant protoplasts. Degenerate oligonucleotide mutagenesis provided single base substitutions at every position in the element and a number of multiple base substitutions. The effects of these changes were determined in transient expression assays with tobacco and maize protoplasts. The majority of single and double base changes had little effect on the activity of the octopine synthase enhancer, but nearly all mutants with more than two base changes had low to essentially no activity. There were five positions where particular single base changes resulted in a 4- to 10-fold loss in enhancer activity. The distribution of these positions within the palindrome was asymmetric. Single base deletions had essentially no activity, demonstrating that the octopine synthase enhancer cannot tolerate internal changes in spacing. We find a strong correlation between mutant phenotype and reduced binding of a protein factor, suggesting that the DNA-protein complex is responsible for the transcriptional enhancement; the functionally active form of the DNA-protein complex probably involves more than a single protein molecule. The mutants exhibit similar phenotypes in protoplasts of both tobacco and maize, implying conservation of the DNA-protein interactions of the ocs enhancer sequence in monocotyledonous and dicotyledonous plants.