Exposing the M.O. of RNA Polymerases, Nature's Gene Decoders


To turn a tape-recorded interview into a newspaper story, journalists must first transcribe the sounds of a voice into words on a page. The cells in our bodies perform a similar task, transcribing information coded in the DNA of our genes into RNA, DNA's chemical cousin. The RNA is then translated into proteins, which perform much of the work in cells. The molecular machines responsible for gene transcription are enzymes called RNA polymerases.

The body contains trillions of cells, virtually all of which have exactly the same set of approximately 25,000 genes. Yet RNA polymerases selectively transcribe only those genes that are appropriate in each cell type, accounting for the differences between liver, nerve, muscle, and other cells. How RNA polymerases know which genes to express in a given cell and under a particular set of conditions has long been a major scientific mystery. Furthermore, the expression of genes in the wrong context can lead to cancer and other diseases.

For these reasons, a thorough understanding of how transcription works holds great promise for developing a broad range of molecular therapies, including potential adult and embryonic stem cell-based treatments. Another application of this knowledge, made possible by the sequencing of the human genome, is tracking gene expression patterns to monitor health and diagnose illness. Researchers are also targeting bacterial versions of the transcription machinery in their efforts to design urgently needed new antibiotic drugs.

Advancing the Case with Technological Improvements
Although biologists have been studying gene transcription by RNA polymerases since these enzymes were first discovered in 1960, details of how the enzymes work at a molecular level have remained elusive. For the most part, previous studies have been limited by the technology available to probe how the transcription process works. A primary reason these enzymes have been so difficult to study is their incredible complexity: RNA polymerases are not just single protein chains, but instead are giant assemblies of a dozen protein chains working together. In addition, they interact with many other accessory molecules that vary according to the cell's particular needs. Without knowing precisely what the many parts look like and how they interact, scientists have been unable to understand the way these intricate molecular machines work to perform their essential biological functions.

As a result of decades of NIGMS funding, scientists have built a substantial base of general knowledge on the transcription process. Technological advances are now providing great opportunities to break new ground in understanding RNA polymerase activity. In particular, NIGMS-sponsored efforts to introduce the application of physics and engineering techniques to biological problems have enabled researchers to examine the inner workings of RNA polymerase and to see single molecules of the enzyme in action.

A case in point is the work of Roger Kornberg, Ph.D., of the Stanford University School of Medicine in Stanford, California. After 20 years of unraveling the biochemistry and genetics of RNA polymerase, Kornberg used X-ray crystallography to obtain a detailed, three-dimensional image of the enzyme that revealed the molecular tasks performed by each of its many parts. This achievement was greatly aided by a new robot system that automates the process of loading and positioning microscopic protein crystals on X-ray diffraction equipment. Jointly funded by NIGMS, the Department of Energy, and NIH's National Center for Research Resources, the robot is one of the high-throughput, or very rapid and efficient, techniques developed by the NIGMS Protein Structure Initiative.

The robot enabled Kornberg and his team to screen 130 crystals in a mere 7 hours without losing a single one. Done manually, this work would have taken many times longer, would have required considerable effort from highly trained scientists, and would have run an increased risk of losing delicate crystals due to human error. Once all of the crystals had been screened, the scientists collected data from the best ones, enabling them to determine the RNA polymerase structure with greater clarity than had ever been achieved before.

Widening the Investigation
All organisms, from bacteria to plants to people, have some form of RNA polymerase. Researchers like Seth Darst, Ph.D., of the Rockefeller University in New York City focus on understanding the RNA polymerases of bacteria. Darst used X-ray crystallography to show how bacterial RNA polymerase pulls apart the two strands of a DNA molecule, threading one strand into a narrow protein cavity and clamping it in place during transcription. The availability of structures from both bacterial and higher organisms provides a guide that may lead to new antibiotic drugs that attack the bacterial version of the enzyme without affecting the human enzymes. An example of an existing antibiotic that targets bacterial RNA polymerase is rifampicin, which is a key component of tuberculosis therapy.

The experiments described above required very small samples of RNA polymerase, less than one thousandth of an ounce. Even so, such samples still contain trillions of molecules, and the results reveal only the average behavior of molecules in this huge collection. Consider the rush of a crowd into an amusement park. Watching the group as a whole, it is impossible to discern the behavior of individual visitors. Similarly, in order to truly understand the mode of action of molecular machines such as RNA polymerases, scientists have been striving to develop methods capable of examining individual molecules in isolation.

Recently, Stanford University's Steven Block, Ph.D., applied techniques from physics and engineering to develop such methods. He observed single molecules of RNA polymerase in action and discovered that the molecules pause at some sites along the DNA for unusually long periods, ranging from seconds to tens of minutes. Block also discovered that when the enzyme detects a mistake during the transcription process, it backtracks, snips out the "typo," and inserts the correct chemical unit. This proofreading capability allows the enzyme to make RNA with remarkable fidelity, reducing the transcription error rate by 100 times or more.

The continuing explosion of insights and technological advances at the interface of biology, physics, and engineering will no doubt yield more evidence of the talents of RNA polymerases. This knowledge, in turn, will deepen understanding of the roles of these enzymes in maintaining health and point to ways of intervening when problems in transcription lead to disease.

To arrange an interview about this research with an NIGMS scientist, contact the NIGMS Office of Communications and Public Liaison at 301-496-7301 or info@nigms.nih.gov.