AMHERST, Mass. – For years scientists have worked to understand molecular events at the initiation of RNA transcription, when special proteins called RNA polymerases, assigned to make a new hemoglobin molecule for example, kick-start the process. Now chemist Craig Martin and molecular biologist Luis Ramirez-Tapia at the University of Massachusetts Amherst have isolated these first steps and provide a new model for how and why it works.
Martin says, “People knew these steps happened, but we’re explaining why they happen and the consequences.” Using a combination of fluorescence imaging techniques in synthetic DNA, the authors propose a new and different model for the energetic changes and structural events at the start of RNA transcription. Results appear in the Journal of Biological Chemistry and are being presented this week, along with the work of chemistry graduate student Satamita Samanta, at the “Post-initiation Events in Transcription” meeting in Virginia.
Understanding what happens as the first 20 or so nucleotides begin the process is important for understanding gene regulation in humans. “These mechanistic details are important for all RNA polymerases in all organisms,” Martin adds. Many human diseases are caused by gene regulation going awry and it is often at the very early phase of RNA transcription that problems arise.
Transcription is part of the process cells use to copy a very specific piece of DNA, to turn genes on and off when needed. Twisted DNA strands, the cell’s genetic library, are made up of thousands of sequential bits called bases. Transcription is often described as RNA polymerases finding a target gene, attaching a cable car or “transcription bubble” where it begins, then munching along to separate the DNA bases by breaking hydrogen bonds as if opening a zipper.
As the RNA separates the DNA strand base by base, it simultaneously begins building the duplicate set by adding new bases on the end of a new RNA strand. But the process is far from smooth. This early phase is characterized by stops and starts in the first 8 to 10 bases known as “abortive cycling,” as if the zipper’s teeth won’t catch, says Martin. Eventually they do catch and transcription moves along the remaining thousands of bases.
“In chemical terms, the protein and DNA bases are held by a strong binding force, keeping them stable. It requires energy to undo that binding,” Martin explains. Where the energy comes from and whether it is chemical or mechanical has been a mystery. Previously, scientists had assumed that the growth of the transcription bubble produced energy and pushing or pressing backwards in a process called DNA scrunching. This is where Martin and Ramirez-Tapia made their discovery.
In his series of experiments, Ramirez-Tapia discovered that every time a base is added onto the end of the new RNA/DNA duplex, it acts like a piston to push the protein off of the DNA such that the promotor is released. The energy comes from adding a new nucleotide onto the end of a piece of RNA.
The UMass Amherst researchers think the protein pushes back on the RNA-DNA rod. Either the piston wins and the initial DNA contacts are released, or the protein disrupts the piston, releasing short abortive RNAs and requiring another try. These initial events happen just once per gene during transcription, and after the initial DNA contacts are released, the whole complex becomes stable and the polymerase will go on for thousands of bases until termination, when its job is complete.
It’s spring-loaded, like tectonic plates that reach a certain tipping point before an earthquake. Ramirez-Tapia’s model can predict the number of bases needed before RNA transcription is triggered.
One technique they used was to work in a mutant DNA strain in which abortive cycling doesn’t occur. As Ramirez-Tapia notes, “The one who doesn’t follow the rule can teach you something.” In each experiment he watched a different base, then put all the different base positions into kinetic models, combined the time courses of three types of experiments and combined multiple observable events.
“We now have a completely new model which wasn’t known before, for why that mutation has the result that it does, and we rationally designed two new mutants to help us test and predict how the process works,” says the biochemist. “So far, the new mutants fit our new model perfectly. Based on the experiments, we feel the new model is correct.”
So why would nature want RNA transcription initiation to work like this? Martin says it makes sense that nature would find a way to couple the “favorable” energetics of nucleotide addition to the “unfavorable” process of disrupting the initial protein-DNA contacts. “We think that in using a not-perfectly-rigid piston as the mechanical coupling, the system doesn’t work perfectly every time, but it works well enough for efficient gene synthesis.”