RNA polymerase. Limit cognate primer for initiation and stable ternary complex formation.

Various lengths of oligoribonucleotides corresponding to regions flanking the initiation site of the A1 promoter of the T7 delta D111 template were examined in order to determine their ability to function as primers of transcription for the DNA-dependent RNA polymerase from Escherichia coli. The oligoribonucleotides which functioned as primers were also examined as precursors for the formation of a stable ternary complex (enzyme X DNA template X oligoribonucleotide product) by systematically extending each primer with one or more specific cognate substrate nucleotide triphosphates. A stable ternary complex (resistant to a salt jump challenge) was formed whenever the primer oligoribonucleotide and augmenting nucleotide triphosphate(s) allowed the formation of the normal third phosphodiester bond of the transcript to occur on the enzyme surface. An oligoribonucleotide (a cognate having the correct base-pairing substituents) containing the preformed third phosphodiester bond does not function as a primer. For example the cognate oligoribonucleotide corresponding to the region flanking the A1 promoter of the T7 delta D111 is: formula; see text The limit primer is the oligoribonucleotide trimer AUC (+1...+3). Other acceptable primers are constructed by adding to this limit primer, cognate bases in the negative registry. The oligonucleotide containing one base added to this limit primer in the positive registry (e.g. AUCG) is completely inactive as a primer. We have also demonstrated these phenomena for the A2 and the A3 promoter of the T7 template.