Stanford researchers solve structure of 'most important protein in biology'

STANFORD -- Stanford researchers have solved the structure of the RNA polymerase protein, one of the pivotal molecules in biology. The polymerase copies genes from DNA to RNA – an essential step in the transfer of information from gene to protein.

'It is arguably the most important protein in biology,' said Roger Kornberg, PhD, professor of structural biology at Stanford University School of Medicine. 'The structure provides the basis for understanding all gene activity in eukaryotic cells,' said Kornberg, whose group’s findings were published in the April 28 issue of Science magazine.

RNA polymerase II is the first apparatus in the production line from gene to protein. Its task is to faithfully copy regions of gene-containing DNA into strands of messenger RNA (mRNA). Once a gene has been copied into mRNA – a process termed transcription – the next step is production of the protein that is coded for by that gene. The protein-making machinery – the ribosome – uses the mRNA as a template for protein production, mirroring how the RNA polymerase enzyme uses DNA as a template for mRNA production.

As the human genome project nears completion, soon the entire DNA sequence of a human being will be revealed. 'But on its own, this information is silent,' said Kornberg. 'RNA polymerase gives it voice.' That is because every cell of the human body contains the same DNA. What makes varied cells – such as blood, nerve and liver cells – differ is the selection of which genes are to be copied by RNA polymerase into mRNA for the eventual production of different proteins in each cell type.

'Transcription of some genes but not others by RNA polymerase is the basis for development of a single cell, the fertilized egg, into a human with many cell types. And the solution of the RNA polymerase structure is necessary to understand this process,' Kornberg explained.

The RNA polymerase enzyme actually consists of 12 separate protein subunits. Using data collected via the method of X-ray crystallography, researchers in Kornberg’s lab constructed a model of how the individual subunits fit together to form the entire RNA polymerase complex.

'This is a machine with moving parts,' said Kornberg. 'We think that one of the important moving parts of this machine is the clamp that swings over the DNA,' said Kornberg.

'Jaws,' 'clamp' and 'funnel' are the names he and his collaborators have given to individual parts of the complex. Regions of three subunits combine to form a pair of pincer-like jaws that trap the DNA near the gene that will be transcribed. The clamp portion of the molecule then swings over the DNA and locks closed, ensuring a tight coupling between the RNA and DNA.

'The affinity of the polymerase for DNA is known to be remarkable,' said Kornberg. 'Transcribing genes of enormous length takes many hours to do. It would be a disaster if [the polymerase] fell off near the end. On the other hand, when it reaches the end it must come off completely,' he said.

Components for the growing mRNA strand enter the machinery through a central pore and funnel. The opening also serves as a waste portal for incorrectly transcribed mRNA. According to Kornberg, the enzyme is believed to be constantly testing the fidelity of the newly produced mRNA. 'The enzyme has the capacity to move forward and backward, like a zipper. It backtracks when proofreading the message,' he said. If an error is found, the faulty message is discarded through the pore before the enzyme resumes production of new RNA.

The technical tour de force that led to his solving the structure of the RNA polymerase molecule has its roots back in the days when Kornberg was a graduate student in the department of chemistry. Based on a discovery he made in the laboratory of Harden McConnell, PhD, professor of chemistry, Kornberg devised a way to make crystals of proteins that were too scarce and too unstable to crystallize using traditional methods. After establishing his own lab at Stanford in the late 1970s, Kornberg and postdoctoral fellows Seth Darst, PhD, Aled Edwards, PhD and others continued developing and refining the methodology. X-ray crystallography is a technique that reveals the three-dimensional structure of proteins. Crystals of a protein are formed when certain chemicals are added to a concentrated solution of the protein. An X-ray beam is then focused on the crystal. As the beams pass through the protein they are scattered by the atoms in the protein, causing a distinctive pattern on a piece of radiographic, or X-ray, film. From this pattern and the intensity of each dot on the film, scientists can figure out how the atoms in the protein fit together and, ultimately, what the protein looks like. X-ray crystallography is one of only two methods commonly used to reveal the 3-D structure of a protein.

The structure of hemoglobin (66 kilodaltons) in the late 1950s was one of the first to be solved by X-ray crystallography. Two of the RNA polymerase subunits (192 and 139kDa) are individually among the largest proteins characterized by crystallography. The entire 12-subunit complex is approximately 500kDa. 'It is the largest protein structure ever determined. It was regarded as a great challenge from the technical standpoint,' said Kornberg.

Members of the Kornberg lab crystallized the RNA polymerase molecule from yeast but previous studies have shown the genetic makeup of the yeast and human proteins to be very similar, leading the scientists to believe that the shape of the two proteins is the same. Yeast and humans are eukaryotic organisms, which carry their DNA packaged in chromosomes inside the cell nucleus. This key feature separates plants, animals and fungi – the group that includes the single-celled yeast – from bacteria. The team worked with yeast because biochemical and genetic studies can easily be done in yeast to confirm and supplement their results.

It took at least five years for the researchers to master the skills to produce crystals of the enormous protein complex, but their troubles did not end with the crystallization. Postdoctoral fellows Patrick Cramer, PhD, Jianhua Fu, PhD and David Bushnell, PhD, worked on the crystals for another five years, learning how to modify traditional methods to cope with the large size of the complex, before the structure was finally revealed. It was December 17, Kornberg remembers, when Cramer computed the final data showing that the problem was solved.

The RNA polymerase enzyme is the focal point of an even larger protein complex that initiates mRNA transcription. Researchers in Kornberg’s lab are now busy solving the structure of the additional 50 proteins that form the larger complex. 'The polymerase stands at the center of this complex of proteins and it’s the part that primarily interacts with DNA and RNA. Any understanding of transcription would have to start with a knowledge of polymerase structure,' said Kornberg.

Cramer and Kornberg are the first and last authors, respectively, of the Science paper. Postdoctoral fellows Averell Gnatt, PhD, and Peter David, PhD, and research assistant Barbara Maier-Davis are co-authors. Researchers from the University of Wisconsin and the University of Toronto also contributed to the paper. The studies were funded by the National Institutes of Health.