T7 Lysozyme Represses T7 RNA Polymerase Transcription by Destabilizing the Open Complex during Initiation

Natalie M Stano,Smita S Patel

doi:10.1074/jbc.m400139200

Abstract

Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation. We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect. Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide). T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide). This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase.

Highlights

The regulation of transcription in bacteriophage T7 RNA polymerase (RNAP)1 as in all RNA polymerases is dependent on the efficiency of each step of transcription, such as binding promoter DNA, binding nucleotides, overcoming abortive synthesis, RNA synthesis, and termination
The kinetics of promoter binding and the formation of the preinitiation open complex were measured in real time using a promoter DNA that was modified with the fluorescent adenine analog 2-AP (Table I)
A crystal structure of the T7 lysozyme-T7 RNAP complex revealed that T7 lysozyme binds to a site distal to the polymerase active site and causes little change in the overall T7 RNAP structure with the exception of the extreme C terminus

Summary

EXPERIMENTAL PROCEDURES

Synthetic DNA and Other Materials—The oligodeoxynucleotides (unmodified and 2-AP-modified) were synthesized by Integrated DNA Technologies (Coralville, IA) and supplied as desalted samples. As described previously [11], the oligodeoxynucleotides were further purified by polyacrylamide gel electrophoresis, electroelution, and ethanol precipitation. The 3Ј-dGTP was purchased from TriLink Biotechnologies (San Diego, CA). Protein—T7 RNAP was overexpressed in E. coli BL21/pAR1219 [12]. The enzyme was purified as described previously [13,14,15] with the exception that the CM-Sephadex separation step was eliminated. The purified enzyme was stored at Ϫ80 °C in 20 mM sodium phosphate, pH 7.7, 1 mM trisodium EDTA, 1 mM dithiothreitol, 100 mM sodium chloride, and 50% (v/v) glycerol.

TABLE I Sequence of the DNA promoters

RESULTS

Promoter synthesis

DISCUSSION