Processivity in Early Stages of Transcription by T7 RNA Polymerase

Craig T. Martin, Daniel K. Muller, and Joseph E. Coleman, Biochemistry 27, 3966-3974, 1988

Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Gralla, J.D. (1980) Biochemistry 19, 3245-3253]. Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces. In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and is less likely following incorporation of GMP into the RNA message. Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded throughout the entire message region. This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling. Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling. Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme. Finally, it is shownthat when transcribing a message of initial sequence GGG…, in the presence of GTP as the sole nucleotide, T7 RNA polymerase synthesizes a ladder of poly[r(G)] transcripts due to a slipping of the RNA messagealong the DNA template. This ladder is largely truncated at a length of 14 bases, providing informationon steric constraints in the ternary complex positioned at +3 on the DNA template.

PMID: 2545254 DOI: 10.1021/bi00434a028