Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters.

The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and -12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerase-promoter complexes.

Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate.

Initiation of RNA synthesis by the phage polymerases is abortive if the concentration of pyrimidine triphosphates is limiting. Under abortive initiation conditions the polymerases repeatedly initiate transcription but produce ribooligonucleotides that terminate just prior to the first occurrence of the limiting substrate. Abortive initiation is most severe if the limiting substrate occurs within the first 8-12 nucleotides of the nascent RNA chain and is particularly evident when UMP is limiting. The formation of stable elongation complexes (as determined by gel retardation experiments) occurs after the synthesis of an RNA product 8-12 nucleotides in length.