19 October 2010

Roger Kornberg – about DNA, RNA, transcription and much more

What does a person have in common with yeast?Text: Anastasia Kazantseva, photo: Ignat Solovey

STRF.ruRoger Kornberg is a professor at Stanford University (USA).

In 2006, he won the Nobel Prize in Chemistry. He could also become a laureate of the prize in physiology or the prize in physics – at the time when these nominations were established, no one expected that the line between sciences would disappear. The award was awarded "for the study of the molecular foundations of eukaryotic transcription" – for recreating the structure of molecules that carry out the transfer of information from DNA to RNA in all nuclear organisms, for example in humans and yeast. On Thursday, October 14, Mr. Kornberg gave a lecture at Moscow State University about his work.

Roger Kornberg: "Today we have studied in detail the structure of three protein complexes,
which are always involved in transcription.
But there are many unexplored substances that occasionally take part in it"

Why transfer information from DNA to RNA? In order to make it easier to understand the importance of Kornberg's research, you need to remember how the implementation of genetic information occurs.

From the point of view of a molecular biologist who sees the world magnified a million times, we all consist of long strands, similar to spaghetti or a washcloth. There is a lot of activity going on in this fibrous world. The spiral, entangled around small lumps of thread, unwinds, a tangle is woven around it, five more coils roll up to this structure, a huge mountain of threads passes from the opposite edge of the spiral to the one that is entangled in a tangle, and this whole structure of threads, shuddering and creaking, begins to build another thread. This is what is called life.

This is what transcription looks like – the synthesis of RNA for subsequent protein production.

The most famous strands are DNA and proteins. Deoxyribonucleic acid is responsible for storing information, you can call it a text or instruction. The sequence of its nucleotides – the gene – uniquely determines which protein will be built. And proteins, long coiled strands of amino acids, carry out absolutely all processes in all cells: right now, muscle proteins allow you to move the cursor, retinal proteins allow you to see letters, nerve cell membrane proteins understand what is written, and mitochondrial proteins, meanwhile, process food and oxygen to provide energy for all these processes.

But the information recorded in DNA cannot be used for protein synthesis directly. The instruction must first be rewritten to an intermediary – RNA, ribonucleic acid. It is this small thread that will come out of the cell nucleus, in which the genes are securely hidden, into the cytoplasm, where you can build anything without fear of damaging the database.  This process of rewriting is called transcription.

RNA is an underestimated molecule, it is almost unknown to the general public, but, most likely, it was the ribonucleic acid molecules capable of storing information and performing active actions that were the first polymers of the first living thing – until they transferred some of their functions to DNA and proteins.

Absolutely all processes occurring in the cell – growth, movement, respiration, differentiation, aging – are possible due to the continuous synthesis of proteins. It, in turn, is possible due to the existence of RNA, which has carried out information about the future structure of the protein from the nucleus. RNA arises as a result of the activity of an enzyme called RNA polymerase, which has read the sequence of nucleotides in a gene and copied it to another carrier. Roger Kornberg studied RNA polymerase, as well as many other proteins involved in transcription.

Especially important for modern biology is the fact that Kornberg studied the transcription of eukaryotes – living beings in which the processes of RNA synthesis and protein synthesis are separated in space and separated from each other by a nuclear envelope. Eukaryotes include protozoa (amoebas, infusoria, etc.), fungi, plants and animals. The process of RNA synthesis in them is much more complicated than in bacteria, it involves a lot more substances. But today, thanks to Kornberg's work, humanity knows a lot about the transcription process. The most important proteins have already been counted, named and studied to the last atom.

Kornberg begins his lecture at Moscow State University with a slide with thanks. It lists the names of several dozen scientists who have studied transcription in eukaryotes. It seems that since the Nobel lecture, the list has become even longer. Talking about the RNA polymerase study, Kornberg refers to his colleagues every two sentences. He attaches great importance to explaining to the audience that modern scientific discoveries are always the result of the efforts of many people and many laboratories. But it was no coincidence that the Nobel Committee chose Kornberg among dozens of specialists: a lot of things that are now included in textbooks were first described in his works.

Transcription difficulties begin with the fact that DNA is very tightly packed. The size of the cell nucleus is several micrometers, and the length of DNA in each cell is measured in meters. In the 70s, Kornberg investigated the first level of DNA stacking. He described nucleosomes – structures of histone proteins around which a DNA double helix is wrapped. It turned out that with such a spatial organization (and the cell constantly stores its hereditary information in this way), no reading occurs and cannot occur either in a cell or in a test tube. This led to the question of what factors separate DNA from histones, what positive regulatory mechanisms make possible the transcription process that the cell needs every second. Others grew out of this question, and their solution required the development of new methods, which then began to be applied in various fields of science.

A chromosome is a very tightly packed DNA molecule.
DNA stacking begins with the double helix (pink)
it twists around a complex of eight histone proteins (green).
The resulting formation is called a nucleosome.

Research on the spatial organization of DNA is actively continuing today, including in the laboratory of Kornberg himself. It has been proven that nucleosomes are very mobile structures and there are many proteins in the cell that can disassemble them, shift them, prevent them from forming and simply modify the chemical structure of histones so that DNA is wound more or less tightly on them. But the main object of Kornberg's scientific interests is the process of RNA synthesis itself according to the instructions laid down in DNA and the proteins responsible for it.

The most important protein – the one that attaches new nucleotides to the emerging RNA molecule – is called RNA polymerase. It was discovered in 1960 by Sam Weiss and Gerard Hurwitz. Interestingly, a year before that, Severo Ochoa and Arthur Kornberg (Roger Kornberg's father) received the Nobel Prize in Physiology or Medicine for the discovery of the mechanisms of biological synthesis of RNA and DNA – their scientific contribution was very significant, but the substance they took for RNA polymerase later turned out to be another enzyme.

Bacteria have one RNA polymerase for the synthesis of all types of RNA. Higher organisms have different enzymes for the synthesis of ribosomal RNA, transport RNA, matrix RNA. Proteins are built on the basis of the nucleotide sequence of the matrix, or informational RNA. It is created by an enzyme called type II RNA polymerase. It was this enzyme that Roger Kornberg studied.

The lecturer explains: in the 80s, three laboratories were engaged in the study of polymerase and other transcription proteins in parallel. All of them worked with histone-free DNA, the transcription of which was recreated in a test tube. Robert Roeder worked with human cells (the immortal HeLa line), Ronald and Joan Conaway (Ronald & Joan Conaway) – with rat cells, and the Kornberg laboratory at Stanford University – with yeast. These studies allowed us to confirm that the transcription process is fundamentally the same in different eukaryotic species. The greatest success in determining the structure and function of transcription complex proteins was achieved by Kornberg. "Ten years, 10 thousand liters of yeast and one graduate student" were needed in order to isolate several grams of proteins from yeast and study their structure.

The most important components of the transcription complex are the RNA polymerase itself and five helper proteins (the main transcription factors are TFIIB, D, E, F and H factor). Polymerase unwinds the DNA double helix, builds RNA and weaves DNA back. But without the main transcription factors, it is not able to recognize the promoter (the part of the gene from which synthesis should begin) and begin transcription.

In addition, there are a huge number of molecules that regulate transcription, that is, determining which proteins the cell will build. Kornberg called the most important protein complex involved in this process a Mediator (this word is capitalized not only out of respect, but also in order not to be confused with mediators – any intermediary molecules). The mediator transmits a signal from the enhancer to the polymerase, and the enhancer is a section of DNA (sometimes very far from the place where transcription takes place) that can perceive signals about the needs of the cell (through transcription factors) and enhance the synthesis of a gene or group of genes. Apparently, this is not the only function of the Mediator: it has already been proven that it participates in transcription in all cases.

In the process of studying the transcription complex, new techniques have been developed to determine the structure of proteins. Kornberg and his team grew two-dimensional protein crystals on lipid membranes to study them under a microscope, and created three-dimensional crystals for X-ray diffraction analysis. These data made it possible to recreate the structure of huge protein complexes with atom accuracy, and such accurate knowledge of the structure allows us to simulate the interaction between DNA, RNA and proteins and understand which functional group is responsible for attaching the correct nucleotide, and which one is responsible for stabilizing the unwound DNA. Kornberg is a person who knows how living matter works. Because protein synthesis is a process by which absolutely all the possibilities of living systems exist: from the movement of the pseudopod of the amoeba to the ability to study the structure of RNA polymerase.

The lecture of the Nobel laureate is coming to an end, the audience has quieted down – biologists are overwhelmed by the greatness of the lecturer, and everyone else is overwhelmed by molecular biology. But the main questions still remain unanswered: what is the practical way out of these complex studies? Is there anything known today about diseases associated with specific mutations in genes encoding transcription factors? Is there, for example, any connection between transcription disorders and the development of cancer?

Roger Kornberg answers in detail:

– Many mutations affecting transcription are known today, and even manifestations of such mutations are known. This can lead, for example, to skin diseases associated with autoimmune reactions. Most mutations are known for factor H. They cause a disorder that manifests itself as hypersensitivity to many factors. But here is a different mechanism of disease development: factor H is involved not only in transcription, but also in DNA repair in case of damage. If it does not work, the rate of accumulation of mutations increases, especially when exposed to some negative factors. Another example is that there are many Mediator mutations. And they are associated with cancer. This is not surprising. A mediator is a regulatory system. This means that a person with a Mediator mutation is predisposed to incorrect gene function. There are many other examples.

The discovery and study of the molecules underlying diseases also means the possibility of developing a treatment. One of the possible ways is to search for small molecules that can be used as drugs, influence the mutant protein and change its work. This area can develop because we know the nature of transcription and know many substances that can affect it. If we know how transcription works, we can imagine what might affect it. An alternative way is to create such molecules that will correct the violation. These will probably be nanostructures. It can work. The information we have is still incomplete, but this question has a right to exist. I think one day it will be done.

Portal "Eternal youth" http://vechnayamolodost.ru19.10.2010

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