Extended Upstream A-T Sequence Increases T7 Promoter Strength

Guo-Qing Tang,Rajiv P Bandwar,Smita S Patel

doi:10.1074/jbc.m508013200

Abstract

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

Highlights

Ance [1,2,3,4]
To investigate whether the AT-rich region upstream of Ϫ17 has an influence on the interactions of the promoter with T7 RNA polymerase (RNAP), we constructed a series of promoter fragments based on the class III promoter ␾9 sequence with its upstream end truncated at different positions from Ϫ27 to Ϫ17 while leaving the consensus sequence downstream of Ϫ17 identical
MP-22, mP1.1B-22, and P-17 appear to have equivalent rates of complex association (TABLE TWO). These results indicate that the AT-rich region upstream of Ϫ17 affects the kinetics of T7 RNAP complex association in a sequence-specific manner

Summary

Introduction

Ance [1,2,3,4]. The structure of T7 RNAP bound to a minimal promoter fragment shows that the specific recognition of T7 promoter involves both base-specific and nonspecific contacts with the 13 conserved promoter base pairs from Ϫ17 to Ϫ5. Promoter ␾3.8 is the only class II promoter with uninterrupted AT runs from Ϫ13 to Ϫ22, and it behaves more like a class III promoter [14, 15] These studies suggest that the AT-rich region upstream of Ϫ17 may play a role in the control of promoter selection during T7 transcription in vivo. To investigate whether the AT-rich region upstream of Ϫ17 has an influence on the interactions of the promoter with T7 RNAP, we constructed a series of promoter fragments based on the class III promoter ␾9 sequence with its upstream end truncated at different positions from Ϫ27 to Ϫ17 while leaving the consensus sequence downstream of Ϫ17 identical. Upstream Sequence Effects on Promoter Strength class II promoter ␾1.1B and a randomly chosen AT/GC-mixed sequence from the Ϫ18 to Ϫ22 position

Methods

Results

Conclusion