Mechanism of Instability in Abortive Cycling by T7 RNA Polymerase

Peng Gong,Craig T Martin

doi:10.1074/jbc.m604023200

Abstract

Abortive transcription, the premature release of short transcripts 2-8 bases in length, is a unique feature of transcription, accompanying the transition from initiation to elongation in all RNA polymerases. The current study focuses on major factors that relate to the stability of initially transcribing abortive complexes in T7 RNA polymerase. Building on previous studies, results reveal that collapse of the DNA from the downstream end of the bubble is a major contributor to the characteristic instability of abortive complexes. Furthermore, transcription from a novel DNA construct containing a nick between positions -14 and -13 of the nontemplate strand suggests that the more flexible promoter reduces somewhat the strain inherent in initially transcribing complexes, with a resulting decrease in abortive product release. Finally, as assessed by exonuclease III footprinting and transcription profiles, a DNA construct defective in bubble collapse specifically from the downstream end exhibits less abortive cycling and little perturbation of the final transition to elongation, including the process of promoter release.

Highlights

Its relative structural simplicity makes bacteriophage T7 RNA polymerase an ideal model enzyme in which to study the enzymology of transcription
By characterizing abortive cycling from DNA constructs with a physical perturbation in the duplex binding region, we show that weakening promoter contacts enhances the stability of initially transcribing complexes throughout the transition to elongation
Extension of the nontemplate strand into the transcribed region to position ϩ3 does not lead to increased instability, indicating that collapse of the bubble from the upstream end does not lead to dissociation of the RNA from the complex

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—His-tagged wild-type T7 RNA polymerase was prepared from E. coli strain BL21, carrying the plasmid pBH161 For comparison of the stability of complexes stalled at positions ϩ6 to ϩ8 on ds and pss constructs (Fig. 1), transcription reactions were initiated and allowed to proceed for 1 min by addition of GTP, ATP, and CTP to a final concentration of 400 ␮M each (without radiolabel), and 2 ␮l of TE buffer containing trace amounts of [␣-32P]ATP was added. For the runoff transcription assay comparing ds and nicked constructs (Fig. 4), 0.25 ␮M each enzyme and DNA were incubated 4 min prior to the addition of NTPs. Sink Challenge Assays—Transcription reactions were carried out as described above except that the NTPs were initially added without radiolabel. During the period of exonuclease digestion, NTP substrates were not reduced sufficiently to perturb the assay

RESULTS

The relatively lower turnover observed from the nicked construct

DISCUSSION