Model for the mechanism of bacteriophage T7 RNAP transcription initiation and termination

Abstract

Characterization of a mutant T7 RNA polymerase (RNAP) that is active on non-promoter templates but has lost the ability to selectively utilize the T7 promoter led to the finding that wild-type T7 RNAP initiates transcription at a high rate on non-promoter templates but that most (~90%) of these initiation events lead to synthesis of dinucleotides only. The anomalously high activity of T7 RNAP on poly(dC) templates (relative to other nonpromoter templates) is due to a reduction in the rate of transcription abortion after dinucleotide synthesis rather than an increase in initiation. Evidence is presented that the transition from abortive to processive transcription is associated with a conformational change in T7 RNAP. The stability of the nascent chain in a ternary complex is shown to increase with increasing chain length in the 2 to 14 base range even when the size of the complementary RNA-DNA hybrid remains constant and small (2 to 3 base-pairs). Two mutant polymerases that show increased release of transcripts during abortive transcription and a proteolytically nicked polymerase that exhibits reduced RNA binding are shown to have reduced ability to read-through a T7 RNAP hairpin U-stretch transcription terminator. Single-stranded nucleic acids are shown to bind more tightly than double-stranded nucleic acids to T7 RNAP. These observations and a large set of published studies on T7 RNAP structure and mechanism are accommodated in a relatively simple model of T7 RNAP transcription initiation and termination in which a T7 RNAP that has initiated transcription is proposed to be capable of assuming two functionally distinct conformations: an abortive conformer characterized by a loose association with the nascent RNA and an inability to translocate along the template; and a processive conformer characterized by the stable retention of the nascent RNA and the ability to process stably along the template. The equilibrium between these two conformations is shifted towards the processive form when the nascent chain binds at a site located at least partly on the T7 RNAP N-terminal domain. This interaction requires that the RNA be more than approximately nine bases and this RNAP-RNA interaction plays a primary role in retaining the RNA within the ternary complex. Transcription termination is a reversal of the abortive to processive switch; hairpin formation disrupts the RNAP-RNA interaction and concomitantly destabilizes the processive conformation. In other words, the nascent RNA is proposed to act as the effector ligand of a non-formal allosteric transition in T7 RNAP that mediates both the switch from abortive to processive transcription and transcription termination.