Researchers at The Rockefeller University have determined the first three-dimensional structure of the cellular RNA polymerase (RNAP), a molecular machine that activates individual genes by transcribing, or reading out, the instructions encoded in their DNA. The structure, published in the Sept. 17 issue of the journal Cell, provides scientists with a model for understanding the RNAPs of higher organisms, including humans.
“We hope that this structure will provide a more detailed framework for interpreting the existing genetic, biochemical and biophysical information, as well as guide further studies aimed at understanding the transcription process and its regulation,” says principle author Seth Darst, Ph.D., associate professor and head of a Laboratory of Molecular Biophysics at Rockefeller.
Molecular biologists often refer to the “Central Dogma,” the roadmap of how information within DNA is transferred to proteins, the building blocks of all living processes. DNA passes information to RNA through a process called transcription, and the RNA carries the blueprints for making proteins. Transcription is orchestrated by a large assembly of proteins called the RNA polymerase. In bacteria, the RNAP comprises four proteins, or subunits. In humans, the RNAP consists of up to a dozen subunits. Because evolution has allowed for genomic similarities among many different organisms, scientists can study the relatively simple structures found in bacteria to understand the complex workings of this machinery in higher organisms, including humans.
Since the late 1980s, Darst’s work focused on the RNAP found in the bacterium E. coli, a well-characterized organism, using a technique called electron diffraction, a method in which electrons are shot at a crystal and the reflected particles provide information about the shape of a molecule. However, electron diffraction only yielded structural information with a resolution of 12 angstroms, meaning that parts of the structure that are much smaller than 12 angstroms can’t be resolved. (An angstrom is the approximate radius of a typical atom.) To obtain a more detailed structure, Darst needed to use a technique called X-ray diffraction, but the RNAP from E. coli did not give good results.
About a year ago, Darst and Gongyi Zhang, Ph.D. (a postdoctoral fellow in the Darst lab, now an assistant professor at the National Jewish Medical and Research Center) decided to look at proteins from thermophiles, organisms that thrive at high temperatures, reasoning that proteins isolated from these organisms would be less flexible and more, characteristics that are desirable for crystallization.
The Rockefeller team chose a bacterium called Thermus aquaticus, a relatively poorly studied organism whose genome has yet to be sequenced. After determining how to purify the RNAP from T. aquaticus,the researchers almost immediately obtained crystals that diffracted X-rays to a nearly 3 angstrom resolution, enough to begin to solve the structure.
However, the researchers still needed more information to solve the structure. They needed to clone and sequence each of the genes whose products make up the subunits. For this, Darst struck up a collaboration with Rutgers University’s Konstantin Severinov, Ph.D., a former postdoc from his lab, to clone and sequence the subunits.
“At every step of the way we expected it would take several months or years, but everything went faster than we thought it was going to,” says Darst. “We got to our current stage in a year.”
The RNAP structure resembles a crab’s claw, with a groove or channel that is the appropriate size for accommodating double-helical DNA. The researchers have proposed a model for how the RNA and DNA are situated in the RNAP during the elongation of the RNA chain.
Darst says that the structure is not complete and needs more work, but even at this stage it’s a big advance for scientists who study transcription. Before the structure was available, scientists studied RNAP by using biochemical techniques, such as mutagenesis, in which individual amino acids in a protein are altered one at a time to yield information about how the protein’s structure relates to its function.
“Now that there is a structure, everything is going to shift from these types of experiments–the blind probing of the structure-function relationship,” says Darst. “Researchers can now look at the structure and design better experiments to really understand what’s going on.”
Along with Zhang, Darst and Severinov, co-authors on the manuscript are Elizabeth A. Campbell, Ph.D., and Catherine Richter, Ph.D., of Rockefeller, and Leonid Minakhin, Ph.D., of Rutgers.
This work was supported in part by a Burroughs Wellcome Career Development Award, a Pew Scholars Award in the Biomedical Sciences, and a National Research Service Award (National Institutes of Health). Campbell was a Kluge Postdoctoral Fellow.