Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA

Wooyoung Kang,Heesoo Uhm,Ja Yil Lee,Kyuhyong Park,Changwon Kang,Kook Sun Ha,Sungchul Hohng

doi:10.1038/s41467-019-14200-3

Abstract

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.

Highlights

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate
Which of the three essential components departs the transcription complex first, RNA polymerase (RNAP) enzyme, RNA product, DNA template, or all together at once? In this study, we used single-molecule fluorescence measurements to primarily examine the dissociations of fluorescently labeled RNA and DNA from unlabeled RNAP, and their post-terminational fates in E. coli intrinsic termination
Our findings include that RNAP on an intrinsic terminator generally releases RNA transcript without departing from DNA template, and the remaining RNAP one-dimensionally diffuses on DNA in both directions and even reinitiates transcription at a nearby promoter

Summary

Introduction

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. Single-molecule experiments using optical tweezers have shown termination mechanisms with RNAP forward hypertranslocation and RNA shearing,[9] which had not been involved in an allosteric mechanism demonstrated by biochemical studies.[10] It is still unknown yet how the complex is broken apart. Our findings include that RNAP on an intrinsic terminator generally releases RNA transcript without departing from DNA template, and the remaining RNAP one-dimensionally diffuses on DNA in both directions and even reinitiates transcription at a nearby promoter

Methods

Results

Conclusion