On the mechanism of oligonucleotide-primed RNA synthesis. I. Model studies with deoxyhomopolymer templates and Escherichia coli RNA polymerase

Abstract

We have studied the effect of temperature, primer chain length and primer concentration on the oligonucleotide-primed transcription by Escherichia coli RNA polymerase. Our experiments with the homopolymer model systems poly(dT) .oligo(A)n, poly(dA) .oligo(U)n and poly(dA) .oligo(dT)n lead to three main conclusions. First, de novo chain initiation on single-stranded templates is preferentially suppressed at higher temperatures. Second, stable annealing of template and primer is neither a prerequisite nor does it stimulate the primer-dependent transcription. Third, formation of the ternary enzyme-template-primer complex is a rate-limiting step in the oligonucleotide-primed RNA synthesis. The maximal rate of primer-stimulated RNA synthesis, moreover, is strongly dependent on the nature of the primer and decreases in the order oligo(A)-primed poly(A) synthesis greater than oligo(U)-primed poly(U) synthesis greater than oligo(dT)-primed poly(U) synthesis. We attribute this to differences in the rate at which the first nucleotide is added to the primer. Raising temperature and primer concentration renders transcription in the model systems almost completely primer-dependent. This can be useful in a transcription approach to DNA sequence analysis.