Reading the genes

Print edition : December 01, 2006

Roger D. Kornberg celebrates his Nobel Prize with his father, Arthur Kornberg, at Stanford University on October 4. Arthur Kornberg won the Nobel Prize for Physiology and Medicine in 1959. - DINO VOURNAS/REUTERS

Roger D. Kornberg wins the Nobel Prize in Chemistry for his study of the molecular basis of eukaryotic transcription.

LIFE in all organisms is essentially the continuous processing of information contained in genes. For this to occur uninterrupted, the information encoded in its DNA (deoxyribonucleic acid) has to be read constantly and correct instructions sent out to body cells. The body cells produce the appropriate proteins that govern various body functions. The central process in all living organisms is, therefore, the "transcription" of genetic information in DNA. If this transcription process is somehow interrupted, say, by the action of certain diseases, as happens with cancer or heart disease, the organism can die.

Roger D. Kornberg of Stanford University School of Medicine, California, was the first to draw a detailed picture of how this molecular process occurs in an important group of organisms called eukaryotes. Eukaryotes, as opposed to bacteria, have a well-defined cell nuclei and include the simple unicellular ordinary yeast common to complex mammalian systems, including humans. The Nobel Prize in Chemistry for 2006 has been awarded to the 59-year-old Kornberg "for his fundamental studies of the molecular basis of eukaryotic transcription".

"The information [in DNA] is one thing," Kornberg pointed out in his first post-award interview, "but its use in the right place at the right time is ultimately decisive. ... Our particular contribution has been [in] analysing the central molecule of the process and discovering the arrangement of the many thousands of atoms that make up the molecule." That molecule is the specific enzyme called RNA-polymerase (RAP), which plays the central role in the transcription process.

The double helical DNA molecule, with its two twisted strands bound to each other in a ladder like structure, lies enclosed within the cell nuclei. DNA is made from four building blocks, T, C, G and A, the "alphabets" of the genetic code. The two strands of the DNA double helix bind in such a fashion that G on one strand is always opposite to C on the other and A in one is opposite to T in the other. The genetic information contained in DNA is determined by the sequence in which these molecular blocks are arranged. This information has to be copied and the instruction carried to protein-producing parts of the cell. The molecule that does this is the messenger-RNA (mRNA).

The RNA molecule also has four building blocks that correspond to those of DNA, which enable it to copy the information. These RNA building blocks exist in solution within the cell. The transcription begins with the DNA being "unzipped" into two strands by RAP with the help of "transcription factors". These assist the polymerase to recognise the "start" site or the "promoter" gene on the DNA, separate the strands and begin RNA synthesis. The building blocks of the naked strand act as a template for this purpose. If the transcription process works, the RNA equivalent of a G inserts itself opposite a C on the strand and the RNA equivalent of T inserts itself opposite an A and vice versa. Step by step the RNA strand is created, which resembles a "negative" of the DNA strand.

The fundamental question for scientists was how this process worked in detail; in order to avoid impairment to the organism the RNA mechanism has to be very specific to ensure a correct reading. In fact, not more than one error in 10,000 can be tolerated. RAP is the key actor in regulating and governing this copying process.

The toadstool death cap causes death because the toxin it carries blocks the function of RAP and slowly destroys the organs. Diseases like cancer, heart ailments and different kinds of inflammations are linked to some interruption in this transcription process. Detailed knowledge about the transcription process can help combat such diseases and also be used to realise the full potential of therapeutic applications of stem cells. All the basic genetic information in body cells is the same. But the great variation between each organ arises from the information that is actually transcribed and processed for protein production. This is the case in the development of stem cells for specific organs.

Kornberg made breakthrough progress in our understanding of molecular transcription and regulation. He applied a combination of advanced biochemical techniques with structural determination to recreate atomic level reconstruction of RAP from baker's yeast (Saccharomyces cerevisiae) in isolation and a number of functionally relevant molecular complexes with template DNA, product mRNA, substrate nucleotides and regulatory proteins. In an ingenious way Kornberg obtained snapshots of the various steps of genetic RAP through X-ray crystallographic methods.. The picture, which was created by Kornberg in 2001, shows RAP in action.

What looks like a bundle of white wires is the RAP enzyme acting as a support for the DNA strand (in blue). The polymerase molecule holds the DNA-strand in the right position during the transcription process and creates a very small "cavity" through which only the right RNA building block can pass, much like a jigsaw puzzle. Once a new building block is inserted correctly, the DNA strand is pushed forward by a small helical spring-like structure (in green) in the polymerase, which flips back and forth as RAP constantly changes its shape. (It is this precise mechanism that the toadstool death cap destroys.) In this fashion, the DNA strand is moved each time into the right position for a new building block to be added to the RNA strand under synthesis.

The extraordinary achievement of Kornberg is that he captured the entire transcription process in pictures. He managed to freeze the process through sheer technical ingenuity to show the RNA strand being built and the position of DNA, RAP and RNA in the process. Normally one sees only pictures of finished biochemical complexes or individual molecules, not a reaction as it takes place. That is the revolutionary aspect of Kornberg's work. He combined his biochemical insight and crystallographic skill to control it at his will.

What Kornberg did was the following. Each time he left out the appropriate RNA building block from the solution he used. When the process of RNA synthesis reached a point when the missing block had to be inserted, the whole process stopped. Kornberg then obtained crystals of these frozen biochemical complexes and produced images of their crystalline structures using X-rays. Starting with such images, the real positions of atoms in the molecules are calculated and appropriate pictures created with the help of a computer. Kornberg has obtained all the pictures since 2000.

Besides the detailed picture of RAP in action, Kornberg's unravelling of the transcription process also includes information regarding the other crystals of the complexes that RAP forms with DNA and RNA, and the transcription factors involved. Eukaryotes have five transcription factors, where prokaryotes, which include bacteria, have only one; it is this that actually makes the transcription process in eukaryotes much more complex.

The transcriptional machinery that Kornberg has unravelled is perhaps the most complicated biochemical process known today. According to Kornberg, it is made up of about 60 protein molecules with each molecule being made up of several thousand atoms. "The remarkable thing, about the machinery," Kornberg says, "is the extent to which it truly functions in the way you and I imagine or think of a machine. It has moving parts, the moving parts function in synchrony, in appropriate sequence and in synchrony with one another. They execute remarkable transitions from one stage or step in the process to the following one and part of the pleasure and the fascination of what we do has been in discovering the mechanics, the inner workings of that machine." According to him, the transcription rate is about 10 DNA or RNA letters per second. It is copied with great fidelity because of inherent mechanisms for proof-reading and correcting errors.

RAP was first discovered in mammalian liver cells, but it was very difficult to work with these cells. Instead, bacteria, which were much easier to use, became the model system to be studied in detail and for a long time it was believed that the transcription process in eukaryotes was similar to that in prokaryotes. The 1965 Nobel award in medicine went to Jacque Monod, Andr Lwoff and Franois Jacob, for their work, among other things, on transcription in bacteria. They found that for the transcription process to begin in bacteria another molecule, called the sigma factor, was necessary. Without this factor, RAP could not begin reading the DNA.

But when scientists looked for the equivalent of the sigma factor in eukaryotes, they found that there was not just one molecule necessary for transcription but five. These complexes play a very important role in regulating and fine-tuning the transcription process. Even after these factors were discovered, isolated and studied, there was no complete understanding of the great variety in shape and function of the eukaryotic cells. That is, one still did not know how certain genes are expressed in blood cells, others in the liver, still others in the brain and so on.

THE TRANSCRIPTION PROCESS as depicted by Kornberg in 2001. RNA-polymerase is shown in white, the bridge helix in green and the active site metal as a pink sphere. The DNA helix is coloured blue while the newly synthesised RNA is in red.-HANDOUT/NOBEL FOUNDATION

One of Kornberg's great contributions was the development of a new model system of unicellular yeast for transcription studies. Yeast, like mammal cells, is a eukaryote, but yeast cells are much easier to manipulate and create a homogeneous material to work with. Yet, Kornberg and associates worked for 10 years to fine-tune the system to make it suitable for transcription studies. Kornberg's perseverance paid off when he could produce the transcription factors for RAP in appropriate form and quantity to create their crystals and investigate. "When we began it was obviously impossible," says Kornberg, "and for much of the time the problems were evidently insuperable. [It was] also a very great investment in time inasmuch as the origins of the work were about 30 years ago and the work began really in earnest towards this objective about 10 years ago."

X-ray crystallography is a particularly important tool in this context because only this technique can reveal the actual spatial configuration of the different components in the transcription system. Kronberg entered the field of transcription when he worked on the structure of chromatin (the nucleo-protein in chromosomes) as a post-doctoral student at the Medical Research Council in Cambridge, U.K., with Francis Crick and Aaron Klug, which involved X-ray crystallography. After his return to Stanford, the focus of Kronberg's research continued to be on understanding transcriptional regulation in eukaryotes.

Using this system of yeast cells, Kornberg discovered yet another molecular complex, which plays an important regulatory role as an on-off switch in the transcription process. DNA includes "enhancers" that bind to specific substances in different tissues. These enable tissue-specific genes transcription to be triggered. For instance, in the liver there is a specific signalling substance that binds to the liver-specific enhancer in DNA and thus causes the transcription of the gene. For functions related to other organs, the liver-specific gene will never be switched on in the absence of the necessary signalling substance.

This regulation mechanism, Kornberg found, required another molecular complex, a kind of "relay" mechanism that transmits signals to switch the transcription on or off. This relay molecule is called a "mediator". It is the fine interplay between tissue-specific substances, enhancers in the DNA and the mediator that enables the great complexity of eukaryotic organisms. The discovery of the "mediator" complex is regarded as a milestone in the understanding of the transcription process. Kornberg's continuing research involves imaging the complete process, including all the transcription factors and the mediator, in full flow using the same techniques of X-ray crystallography.

Kornberg has continued to investigate yeast cells because the similarity of the transcription machinery between yeast cells and higher organisms such as plants and mammals, including humans, is striking. "When we began our work, we made the decision to focus on a simple, unicellular eukaryote, baker's yeast, and the decision at that time was one of uncertain wisdom because it did seem from the available information [that] there might be profound differences between this fundamental process in yeast and, for example, in human cells. [But] the similarity goes far beyond anything we had anything we had anticipated," Kornberg said. This is not only the case for polymerase; the large cast of crucial molecules that assist and regulate the transcription process are virtually the same in both yeast and higher cells.

Arthur Kornberg, his father, received the Nobel Prize in Physiology and Medicine in 1959. He described how genetic information is transferred from one DNA molecule to another, specifically from a mother cell to daughter cells.

Now, Kornberg has described how genetic information is read and processed to make proteins.

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