Protein machinery

Print edition : November 20, 2009

Venkatraman Ramakrishnan of the Medical Research Council Laboratory, U.K.-ALASTAIR GRANT/AP

THE deoxyribonucleic acid (DNA) molecules wrapped up in every cell of every organism, from a bacterium to a plant to a human being, in the form of chromosomes contain the blueprint of life. But what is life? In a reductionist paradigm, it is an aggregate of the organisms actions its looks, functions and behaviour. Clearly, the central process of life is how the chemical information coded in the DNA is translated into lifes various biological processes, each one of which is controlled by one or many of the tens of thousands of proteins that the body cells produce. Elaborate biochemical machinery that has been evolutionarily conserved over millions of years achieves this to great precision. It turns the information contained in the thousands of genes characteristic of the organism its genome into these thousands of functional proteins, such as haemoglobin that carries oxygen, insulin that controls sugar levels in the blood, antibodies that defend against infections or melanin that determines the skin colour.

The DNA molecule is made up of a string of four variously arranged, smaller, basic molecular building blocks called nucleotides A (adenine), C (cytosine), G (guanine) and T (thymine). The three-dimensional structure of the DNA is a double-stranded helix, where A is paired with T and C with G. The sequences in which these nucleotides are strung together code for proteins that perform the various biological functions in the body. But the DNA, comprising only four small molecules, alone cannot do this. This process is mediated by another nucleic acid molecule called ribonucleic acid (RNA), which is also made of four building blocks, with T, however, being replaced by U (uracil). Most of the RNA is found in the cytoplasm (Figure 1) located outside the nucleus on the other side of the nuclear envelope. It is also in the cytoplasm, away from the nuclear DNA, that proteins get produced by a molecule called the ribosome. The ribosome consists of proteins and RNA molecules called ribosomal RNA (or rRNA).

So the mechanism is the following. The genetic information in the genes contained in the DNA is copied on to an RNA molecule called messenger-RNA (mRNA). This transcription of DNA into mRNA is achieved by an enzyme called RNA polymerase. Roger Kornberg got the Nobel Prize for medicine in 2006 for elucidating this (Frontline, December 1, 2006). The mRNA moves outside the nucleus and is caught by the ribosome. The mRNA is the blueprint for producing proteins; the ribosome reads the nucleotides on the mRNA in triplets, which are called codons. The ribosome actually uses another RNA molecule, called the transfer-RNA (tRNA), to read. At one end of the tRNA, there is a nucleotide anticodon triplet that pairs with a matching codon on the mRNA in the ribosome. This results in the production of an amino acid as coded by the codon (Figure 2).

One or several of these codons code for an amino acid and the sequence of amino acids determines the protein. The first codon that scientists discovered was UUU, which is translated by the ribosome as the amino acid phenylanine. There are 64 different codons and 20 different amino acids. The genetic information in the DNA is, of course, preserved by the chromosome replication mechanism carried out by DNA polymerase so that each daughter cell receives one genome copy at every cell division and the proteins appropriate to a given cell type continue to be produced. This then is the central dogma of molecular biology, as Francis Crick termed it in 1970:

Ada Yonath of the Weizmann Institute of Science in Israel.-DAN BALILTY/AP

DNA (gene) RNA (mRNA) Protein (peptide sequence).

The above picture, however, is only schematic. Unfortunately, James Watson, who unravelled the double helical structure of the DNA along with Crick and Maurice Wilkins in 1953, noted in 1964: We cannot accurately describe at the chemical level how a molecule functions unless we first know its structure. It took three and a half decades for the deciphering of the ribosome structure, which tells you how each atom is located in the molecule.

Thomas A. Steitz of Yale University in Connecticut, U.S.-LUCAS JACKSON /REUTERS

This years Nobel Prize in Chemistry has gone to 57-year-old Venkatraman (Venki) Ramakrishnan, an American of Indian origin at the Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, United Kingdom; 69-year-old Thomas A. Steitz of Yale University and Howard Hughes Medical Institute, U.S.; and 70-year-old Ada E. Yonath of the Weizmann Institute of Science, Israel, for unravelling the ribosome structure and how it functions at the atomic level. For determining the ribosome structure, all three used the technique of X-ray crystallography and mapped the position of each one of the hundreds of thousands of atoms that the ribosome consists of.

Over and above its importance in the scientific understanding of lifes different processes, the knowledge of the ribosome structure has also become useful in medicine. Most of the known antibiotics target the bacterial ribosome and work by blocking its function, and all the three Nobel laureates have provided a detailed understanding of how antibiotics interact with the ribosome through three-dimensional models of this interaction. This has today enabled structure-based design and development of new and more effective antibiotics that can overcome the bacterial ability to mutate to drug-resistant forms.

During the 1970s, generating X-ray crystallographic structures of the ribosome was considered extremely difficult, if not impossible, because the ribosome is a large 2.5 MDa molecule and does not have symmetrical properties that facilitate crystallisation and structure determination. Ada Yonath, however, decided to venture into that uncharted territory despite the scepticism of many people. In X-ray crystallography, X-rays are beamed at the crystal of the molecule whose structure is to be determined and the scattering of the X-rays by the atoms (diffraction) is captured in detectors, a photographic film-based one in the early days, which was later replaced by more sophisticated digital Charge Coupled Device (CCD) cameras. Information about how the atoms, the scattering centres, are arranged in the crystal lattice can be gleaned by analysing the diffraction pattern, the pattern of black spots formed by the spreading X-rays on the film. Basically, the position and intensity of each spot is measured, from which what is called the electron density is calculated. From the electron density maps, the molecular models of the ribosome structure are obtained.

For the technique to yield accurate results, the crystal lattice has to be nearly perfect; that is, the crystal should have a fundamental structural unit of atoms that is repeated with a precise periodicity. The more complex or larger the molecule, the harder is this task of producing high-quality crystals. And the ribosome is one of the most complicated RNA complexes. It has two parts, the large subunit (50S) and the small subunit (30S), with molecular weights of about 1,500,000 Dalton (Da) and 800,000 Da. In a human ribosome, the small 30S subunit consists of a large RNA molecule (containing 1,600 nucleotides) and 20 different proteins. The large subunit (50S) consists of 33 different proteins and two RNA molecules, one with about 2,900 nucleotides and the other, small one with about 120 nucleotides. Thus, each subunit comprises thousands of nucleotides and thousands of amino acids. These nucleotides and amino acids are, in turn, made up of thousands of atoms. It is the daunting task of locating each of one of the atoms in the ribosome structure that Ada Yonath set out to do.

Ada Yonath chose the bacterium Geobacillus stearothermophilus that lives in warm springs and survives in such harsh conditions where temperatures can go up to 75C. The rationale for the choice was that, given the requirement of a very stable ribosome for forming better crystals, a robust bacterium should work. Ada Yonath obtained her first crystals in 1980. Although these were far from perfect, it was indeed a breakthrough achievement. But it was not enough to obtain a three-dimensional image of the ribosome where the location of each atom was clear. She had to wait for another 20 years before that could be achieved.

Ada Yonath tried many new things, like stabilising the crystals by freezing them in liquid nitrogen at 196C. She also tried to obtain crystals from another equally hardy organism, Haloarcula marismortui, which lives in the highly saline environment of the Dead Sea. Ada Yonaths work during the 1980s was instrumental in obtaining robust and well-diffracting ribosome crystals that eventually led to high-resolution structures of 30S and 50S subunits; by 1990, it became clear that the crystals had sufficient quality for the ribosome structure to be mapped. This led to other scientists such as Thomas Steitz and his collaborators from Yale University, and Venki Ramakrishnan and his collaborators from MRC, Cambridge, entering the scene.

There was still, however, a crucial unsolved problem. It was what is called the phase problem in X-ray crystallography. Now light (X-ray) is a wave that has both amplitude and a phase. However, since measurement involves only detecting the light intensity at every dot, this phase information, which carries important information about the location of the atoms in the crystal, is lost. This phase information has to be recovered for determining the three-dimensional structure from a diffraction pattern.

A method used by X-ray crystallographers to recover the phase information is to modify the crystals by adding organic clusters of heavy metals, such as mercury, by soaking the crystals with suitable heavy atom solution. These heavy atoms function as a kind of road marker. Specifically, Ramakrishnan and Steitz used clusters containing tungsten and osmium. The heavy atoms attach to the surface of the crystals ribosomes. To protect the crystallised ribosomes from radiation damage in the course of bombarding them with X-rays, they were flash-frozen to about 175C. By comparing the diffraction patterns with and without the heavy atoms, the phase information can be retrieved. However, because of the large size of the ribosome molecule, the problem remained intractable. Steitz finally solved the problem by using the electron microscope images of the molecule obtained by Joachim Frank, a specialist in electron microscopy. Even though their resolution was not good enough to visualise individual atoms, they could be used to determine the orientation of the ribosomes within the crystal. This information, together with the diffraction pattern from the heavy atom-soaked crystal, finally yielded the phase angle.

Steitz published the first crystal structure of the ribosomes large subunit in 1998, which looked like a dim photograph. It had a resolution of 0.1 nanometre (1 nm is a billionth of a metre). Though individual atoms could not be seen, the ribosomes long RNA molecules were visible, which was certainly a major breakthrough. The problem now, after the solution of the phase problem, was that of improving the image quality and resolution so that atomic locations could be interpreted properly. Progressive improvement in crystals also meant higher and higher resolution. Ada Yonath, Steitz and Ramakrishnan succeeded in doing so almost simultaneously in the year 2000 as if it was a magic year for ribosome research. Steitz managed to obtain the structure of the large subunit from Haloarcula marismortui. Yonath and Ramakrishnan obtained the structure of the small subunit from Thermus thermophilus. The ribosome functionality was now mapped at the basic atomic level.

The primary role of the large subunit 50S in the ribosome is to synthesise new protein. It triggers the formation of the peptide bond between amino acids. In a single ribosome, as many as 20 peptide bonds are formed every second. But imaging every step of this process is very difficult because, for one, it occurs at the atomic level and, two, it takes place at a fantastic speed. Steitz, however, managed to freeze different moments of bond formation. He crystallised the large subunit with molecules similar to those that are involved in the formation of the bonds. With the help of these structures, it has been possible to determine which of the ribosomes atoms are important to the reaction and how the reaction occurs.

The ribosome, it has been seen, rarely makes errors in translating the DNA/RNA language into the protein language and the small subunit seems to perform the role of checking for accuracy in the translation. An error in the addition of an amino acid in the protein assembly, for example, can result in its functionality being lost or the protein function being changed entirely, which could even be harmful. The selection of the amino acid depends on the base pairing process between mRNA and tRNA (Figure 2). However, this alone is not sufficient to explain the fidelity in the ribosomes action.

Ramakrishnans structures of the 30S subunit of the ribosome have been crucial for understanding how the ribosome achieves this precision. Nucleotides in the 30S subunits rRNA, he found, served as a kind of molecular ruler to check if the translation process was accurate. To put it simply, they measure the distance between the codon in mRNA and the anticodon in tRNA. If the distance is incorrect, the RNA molecule falls off the ribosome. The ribosome double-checks for accuracy by using this molecular ruler twice. This ensures that the error in translation is one per 100,000 amino acids. If the high-resolution 50S structures were instrumental in the clarification of how ribosomes catalysed the peptide bonds, the high-resolution 30S structures of Ramakrishnan and collaborators have enabled a simple and coherent explanation for the hitherto poorly understood phenomenon of accuracy of codon reading during mRNA translation.

Once high-resolution structures of both the subunits, 50S and 30S, were obtained an important finding was that the peptidyl-transferase centre, where peptide bond formation is actually catalysed, seemed to lack ribosomal protein components. It was, in fact, found that there was no visible peptide chain within 2 nm of the transferase centre. This has been regarded as proof of the hypothesis that the ribosome is, in fact, a ribozyme; that is, an enzyme whose catalytic action is derived entirely from its RNA and not its protein components. This result gives added support to the widely emergent view that the present biochemical world, in which proteins carry out the vast majority of biological functions, was preceded by an RNA world where RNA not only was an information carrier but also performed specific tasks. More recent work has, however, shown that both the ribosomal RNA and the ribosomal protein catalyse the peptide bond formation.

The achievements of the three laureates, as described above, were made possible by (i) successful production of the crystals of the ribosomes and their subunits, which for many years had been a major hurdle, and (ii) by the availability of shortwave penetrating X-rays, which are required for high-resolution images of large structures. The penetrating X-rays are generated in large ringed machines called synchrotrons, in which particles such as electrons move in circular paths at high speeds while radiating a wide spectrum of light, including X-rays of high intensity.

By shining X-rays, which are tapped through ports in the accelerator ring, through crystallised molecules and analysing the diffraction patterns, the three-dimensional structures are determined.

The Nobel Prize for scientists who used intense synchrotron-based light sources for their studies of large molecular structures such as ribosomes is also a recognition of the importance of synchrotron X-ray crystallography as a technique for biologists to unravel structures of large molecules (Figure 3). Indeed, the X-ray crystallographic studies of ribosomes became possible only with the advent of synchrotron radiation sources. Steitz and Ramakrishnan used the Advanced Photon Source (APS) at the Argonne National Laboratory; the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory, New York; and the Stanford Synchrotron Radiation Laboratory at Stanford University, California. Ada Yonath, a good part of whose work was also carried out when she led a group at the Max Planck Institute for Molecular Genetics in Berlin during 1989-2004, largely used the Cornell High Energy Synchrotron Source (CHESS) at Cornell University, New York, and also the Deutsch Elektronen Synchrotron (DESY) in Hamburg.

In recent years, structure-based drug design (SBDD), where high-resolution images of drug targets and their resistant mutants are used to create new drugs, has emerged as a promising area of application of basic biochemical research in medicine. The ribosome, as mentioned earlier, is the target for about 50 per cent of all antibiotics to date. The availability of high-resolution structures of both the ribosome subunits thanks to this years Nobel winners has opened a large avenue for SBDD of new and effective antibiotics that can beat the antibiotic-resistant mutants of bacterial pathogens.

Since ribosomes are ancient, ribosomes of bacteria and humans have diverged considerably over the course of evolution. As a result, antibiotics can work. An antibiotic binds to a specific part of the RNA in a bacterial ribosome and distorts it, preventing it from functioning normally, and thus kills the bacteria. Because of the divergence between human and bacterial ribosome, an antibiotic can act on bacteria without seriously affecting humans. For instance, many types of antibiotic drugs bind to the peptidyl transferase centre of the 50S subunit of the bacterial ribosome. Some of them block the exit tunnel through which the growing protein leaves the ribosome. Other antibiotics prevent the formation of the peptide bond between amino acids or corrupt the translation from DNA/RNA language into protein language.

Antibiotic resistance arises, for example, because of mutation of a single nucleotide base, from an A to a G in the site where antibiotics bind to the ribosome. Armed with ribosome structures, one can now see structural alterations that result in antibiotics being bound to the ribosomes with different sensitivity because of mutation. One now understands why mutation has the effect that it does. According to Steitz, the mutant G has an amino group that pokes into the centre of the antibiotics ring structure, causing it to back off the ribosome by a fraction of a nanometre or so. Several companies, including the one founded by Steitz, called Rib-X Pharmaceutical, are developing a new class of antibiotics that are also undergoing clinical trials.

The groundbreaking discoveries of Ada Yonath, Steitz and Ramakrishnan have thus helped clarify long-standing and fundamental questions on protein synthesis. This, in fact, is an expanding area of research today with studies on a new-generation, functional crystal complexes of the ribosome. Besides, their contributions have also proved to have far-reaching implications for medicine and human health.

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