Bacterial transcription initiation gone awry: backtracking and pausing that lasts for durations comparable to the bacterial lifespan
Date:
Lecture / Seminar
Time: 14:00-15:00
Title: Special Guest Seminar
Location: Max and Lillian Candiotty Building
Lecturer: Dr. Eitan Lerner
Organizer: Department of Immunology and Regenerative Biology
Details: UCLA
Abstract: DNA transcription follows a chain of events: initiation, elongation and terminat ... Read more DNA transcription follows a chain of events: initiation, elongation and termination, in which initiation usually the slowest. This is mainly due to the process of RNA-polymerase (RNAP) proper binding to the promoter region on DNA and formation of the open transcription bubble, but also due to many failed attempts of the initially-transcribing complex (ITC) to escape the promoter region and transition to elongation. The latter involves multiple polymerization rounds of short transcript that are depleted from the complex after RNAP aborts a transcription trial to try again (abortive initiation). Traditionally, each round of abortive initiation was thought to be rapid. Using single-molecule FRET as well as magnetic tweezers nanomanipulation tools we have recently discovered an abortive initiation intermediate in which a short transcript on its way to be depleted, stabilizes the complex in a unique conformation with blockage of the nucleotide entry channel (the secondary channel). Even more intriguing was the fact that this paused-backtracked initiation intermediate was stabilized for ~4600 s. In addition, using single-molecule FRET measurements of multiple distances, we show that this long-lived paused-backtracked intermediate is associated with a conformation in the DNA transcription bubble different than any existing determined structure of the bacterial transcription initiation complex. Additionally, the initiation complex in this intermediate state avoids inhibition by the antibiotic molecule Rifampicin, for which there exist many different antibiotic-resistant mutants of RNAP. Therefore, it is important to understand how to stabilize this long-lived paused state as an antimicrobial strategy. This requires further structural determination, and because this intermediate state is heterogeneous, hence very hard to resolve using traditional structural biological techniques, we will discuss ways to resolve the possible structure through hybrid/integrative structural techniques, combining single-molecule spectroscopy and coarse-grained simulations. These findings open a new avenue in studying the mechanism of bacterial transcription initiation as well as new molecular therapeutic routes.Close abstract
