Effects of discontinuities in the DNA template on abortive initiation and promoter escape by Escherichia coli RNA polymerase

Abstract

Using singly gapped or nicked templates containing the T7A1 promoter, we have measured several kinetic parameters related to the process of transcription initiation by Escherichia coli RNA polymerase, confirming and extending previous results using a population of randomly gapped templates. A reduced probability of transcript abortion at RNA lengths of 6 and 7 nucleotides and a lower ratio of abortive to productive initiation events was observed for some discontinuous templates, consistent with models attributing abortive initiation to the accumulation of strain in the initiating complex. The effect of DNA discontinuity on abortion of shorter RNA transcripts (2-3 nucleotides) was less pronounced; abortion at these short chain lengths may primarily be attributed to the low stability of the RNA-DNA hybrid. Certain discontinuities had significant effects on the intrinsic catalytic capacity of the open complex and also on the partitioning between productive and unproductive complexes, suggesting that subtle changes in the conformation of the open complex can profoundly affect its function. The rate and efficiency of promoter escape were not correlated with the stability of the open promoter complex despite previous suggestions to the contrary. We conclude that the stability of the open promoter complex is only one of several factors that contribute to the overall rate of promoter escape. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.