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.
Highlights
Introduction of a nick into theDNA had varying effects on the rate of dinucleotide synthesis
In previous work [12] we explored the roles of specific template nucleosides in the process of promoter escape by E. coli RNA polymerase at the T7 A1 promoter
We depict the ability of open promoter complexes formed on templates gapped at various nucleoside positions in comparison to those formed on an intact template to escape the promoter and form a stable ternary elongation complex (TEC)
Summary
RNA Polymerase—E. coli RNA polymerase (RNAP) with a His tag at the C terminus of the Ј subunit was purified from strain RL712 After EPo formation and at various times after heparin addition, a 5-l aliquot of the reaction mixture was removed and mixed with sucrose loading buffer (60% sucrose, 0.01% bromphenol blue, 0.01% xylene cyanol) and immediately loaded onto a running native 5% polyacrylamide gel (acrylamide to bisacrylamide ratio of 37.5:1) at 20 °C. At various times after heparin addition, transcription was initiated by adding a substrate mixture containing 50 M each of [␥-32P]ATP (750 cpm/pmol), CTP, UTP, and GTP. Samples were removed, mixed with an equal volume of formamide loading buffer, and subsequently analyzed by electrophoresis on a 23% denaturing gel (19:1 acrylamide:bisacrylamide). The abortive probability, defined as the probability that a transcript will abort rather than extend to the position, can be calculated using the following equation [21], where Pi is the probability of aborting transcription at the ith position, and Xi is the percent yield of transcript at the ith position at the 10-min time point
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