Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences.

Masahiko Imashimizu,Hiroki Takahashi,Yuji Tokunaga,David B Lukatsky,Nobuo Shimamoto,Ariel Afek

doi:10.3390/biom10091299

Abstract

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on the sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences, when enriched with thymine bases, contain the signal for inducing abortive transcription, whereas certain repetitive sequence elements embedded in promoter regions constitute the signal for inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we suggest that repetitive sequence elements within the promoter DNA modulate the nonlocal base pair stability of its double-stranded form. This stability profoundly influences the reaction coordinates of the productive initiation via pausing.

Highlights

In bacteria, transcription at a promoter is initiated by σ factor that forms a holoenzyme by binding to RNA polymerase (RNAP) core enzyme
We have previously developed the RNase-footprinting followed by NET-seq (RNET-seq) method to identify the complexes that are paused during transcription elongation in E. coli wild type (WT)
All RNAPs including those associated with the fragmented double-stranded DNA (dsDNA) and their 5 -truncated nascent RNAs were immobilized on Ni2+-NTA beads via the histidine-tagged β’ subunit and washed

Summary

Introduction

Transcription at a promoter is initiated by σ factor that forms a holoenzyme by binding to RNA polymerase (RNAP) core enzyme. E. coli promoters targeted for transcription initiation by σ70 holoenzyme have been characterized by having two consensus motifs approximately 10 and 35 bases upstream of the transcription start site (TSS). These motifs consist of a TATAAT (−10 box) and a TTGACA (−35 box), conserved in the promoters with high binding affinity to σ70 holoenzyme [1,2,3]. Biologically functional promoters with high transcriptional activities usually do not have the full consensus motifs, but rather have nonlocal sequence signatures across the overall promoter region [4] The reason for this is partially due to the need for RNAP to escape the promoter [5] but it has not been fully elucidated with respect to the regulatory mechanism of transcription initiation

Methods

Results

Discussion

Conclusion