Switching gears: novel platform spills secrets of transcription’s speed regulation
Thanks to a first-of-its-kind platform, we are starting to understand how the molecular engine responsible for transcription in mammals controls its speed.
A team of researchers from The Rockefeller University (NY, USA) and Fudan University Shanghai Cancer Center (Shanghai, China) has invented a novel single-molecule platform to illuminate the intricacies of mammalian DNA transcription, with far-reaching implications throughout biology, including cancer and aging research.
Transcription elongation by RNA polymerase II (Pol II) – the enzyme complex responsible for transcribing DNA into mRNA in eukaryotes – is an essential part of gene expression. During this process, the speed at which the protein complex moves along the DNA template is not constant and is governed by several elongation factors.
In metazoans, we know that Pol II pauses after assembling the initiation complex at the promoter and transcribing 30–60 nucleotides of RNA. At this point, elongation factors such as P-TEFb, DSIF and PAF1C initiate the release of Pol II and kickstart a period of rapid transcription, before the molecular machine slams on the breaks and decelerates toward the 3′ end of the gene to allow for the termination of transcription.
Dysregulation of elongation has been linked to disease and aging; however, the exact regulatory mechanisms that underpin it remain incompletely understood, especially for higher eukaryotes. Research on the mammalian elongation complex has thus far been limited by a lack of appropriate techniques; those techniques used could not illuminate the contributions of individual proteins nor capture the complexity of eukaryotic regulatory mechanisms in single-molecule studies in simpler organisms.
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To combat this, the team built a single-molecule platform to watch mammalian transcription elongation in real time. They reconstituted the mammalian elongation complex using purified proteins assembled on a synthetic nucleic acid scaffold containing an RNA primer with its 5′ end fluorescently labeled and a DNA template that was biotinylated at either end.
The construct was then tethered between a pair of streptavidin-coated beads and visualized on a C-Trap instrument that combines dual-trap optical tweezers and single-molecule fluorescence microscopy.
Combining biochemistry, single-molecule imaging and computation, the researchers were able to see Pol II in action, following along as it accelerated and paused, and determine the role of each elongation factor responsible for its kinetics.
“This is the first time we’ve been able to see mammalian Pol II move at a physiological speed in real time and, because we labeled the various elongation factors, we were also able to measure their binding kinetics during active transcription,” first author Yukun Wang explained.
The team identified a hierarchy of several key elongation factors – P-TEFb, DSIF, PAF1C, SPT6 and RTF1 – which act synergistically to achieve optimal elongation activity.
“What’s really striking is how this machine functions almost like a finely tuned automobile,” added corresponding author Shixin Liu. “It has the equivalent of multiple gears, or speed modes, each controlled by the binding of different regulatory proteins. We figured out, for the first time, how each gear is controlled.”
As well as shedding light on how this regulatory mechanism may be involved in disease and opening doors for therapeutic design – P-TEFb, for example, is considered a promising drug target for leukemia and solid tumors – the platform proves that single-molecule visualization is possible in a fully reconstituted mammalian system. As such, there are a multitude of potential applications, in biology and beyond.
“Anything that involves navigation in space and changes in speed could potentially use this software,” prophesized Joel E. Cohen, an author on the paper.
