One gene one enzyme modern interpretation. Gene expression during protein biosynthesis. Regulation of gene expression in pro- and eukaryotes. Hypothesis "one gene - one enzyme", its modern interpretation. PS4 launched, Xbox One on the way: one on one or two

4.2.1. One gene, one enzyme hypothesis

First research. After in 1902 Garrod pointed out the connection of a genetic defect in alkaptonuria with the body's inability to break down homogentisic acid, it was important to elucidate the specific mechanism underlying this disorder. Since then it was already known that metabolic reactions are catalyzed by enzymes, it could be assumed that it was the violation of some enzyme that leads to alkaptonuria. Such a hypothesis was discussed by Driesch (in 1896). It was also expressed by Haldane (1920, see) and Garrod (1923). Important stages in the development of biochemical genetics were the work of Kuhn and Butenandt on the study of eye color in the mill moth. Ephesia kuhniella and similar studies by Beadle and Ephrussi on Drosophila(1936). In these pioneering works, insect mutants previously studied by genetic methods were selected to elucidate the mechanisms of action of genes. However, this approach did not lead to success. The problem turned out to be too complicated, and in order to solve it, it was necessary:

1) choose a simple model organism convenient for experimental study;

2) to look for the genetic basis of biochemical traits, and not the biochemical basis of genetically determined traits. Both conditions were met by Beadle and Tatum in 1941 (see also Beadle 1945).

Beadle and Tatum model. Their article began like this:

“From the point of view of physiological genetics, the development and functioning of an organism can be reduced to a complex system of chemical reactions that are somehow controlled by genes. It is quite logical to assume that these genes ... either act as enzymes themselves, or determine their specificity. It is known that genetic physiologists usually try to investigate the physiological and biochemical foundations of already known hereditary traits. This approach made it possible to establish that many biochemical reactions are controlled by specific genes. Such studies have shown that enzymes and genes have the same order of specificity. However, the scope of this approach is limited. The most serious limitation is that, in this case, hereditary traits that do not have a lethal effect and, therefore, are associated with reactions that are not very important for the life of the organism, fall into the field of view of researchers. The second difficulty ... is that the traditional approach to the problem involves the use of outwardly manifest signs. Many of them are morphological variations based on systems of biochemical reactions so complex that their analysis is extremely difficult.

These considerations led us to the following conclusion. The study of the general problem of genetic control of biochemical reactions that determine development and metabolism should be carried out using procedure opposite to the generally accepted: instead of trying to find out the chemical basis of known hereditary traits, it is necessary to establish whether genes control known biochemical reactions and how they do it. The ascomycete neurospore has the properties that make it possible to implement this approach and, at the same time, serves as a convenient object for genetic studies. That is why our program was built on the use of this particular organism. We proceeded from the fact that X-ray exposure causes mutations in the genes that control certain chemical reactions. Suppose that in order to survive in a given environment, the organism must carry out some kind of chemical reaction, then a mutant deprived of such an ability will turn out to be unviable under these conditions. However, it can be maintained and studied if grown in a medium to which the vital product of a genetically blocked reaction has been added.”

4 Action of genes 9

Rice. 4.1. Scheme of the experiment for the detection of biochemical mutants of neurospores On a complete medium, mutations induced by X-rays or ultraviolet do not interfere with the growth of the fungus. However, the mutant does not grow on minimal medium. When vitamins are added to the minimal medium, growth is restored When amino acids are added, there is no growth Based on these data, it can be assumed that the mutation occurred in the gene that controls the metabolism of the vitamin The next step is to identify the vitamin that can restore normal function The genetic block was found among the reactions of vitamin biosynthesis .

Next, Beadle and Tatum describe the design of the experiment (Figure 4.1). The composition of the complete medium included agar, inorganic salts, malt extract, yeast extract and glucose. The minimal medium contained only agar, salts, biotin, and a carbon source. The mutants that grew on the complete medium and did not grow on the minimal medium were studied in the most detail. In order to establish the compound, the synthesis of which was impaired in each of the mutants, individual components of the complete medium were added to the minimal agar.

In this way, strains were isolated that were unable to synthesize certain growth factors: pyridoxine, thiamine, and para-aminobenzoic acid. These defects have been shown to be due to mutations at specific loci. The work marked the beginning of numerous studies on neurospores, bacteria and yeasts, in which a correspondence was established between the "genetic blocks" responsible for individual metabolic steps and specific enzyme disorders. This approach has rapidly evolved into a tool for researchers to uncover metabolic pathways.

The hypothesis "one gene - one enzyme" has received strong experimental confirmation. As the work of subsequent decades showed, it proved to be surprisingly fruitful. The analysis of defective enzymes and their normal variants soon made it possible to identify a class of genetic disorders that led to a change in the function of the enzyme, although the protein itself was still detectable and retained immunological properties. In other cases, the temperature optimum of enzyme activity changed. Some variants could be explained by a mutation that affects the general regulatory mechanism and, as a result, changes the activity of a whole group of enzymes. Such studies led to the creation of the concept of regulation of gene activity in bacteria, which included the concept of the operon.

10 4. Action of genes

The first examples of enzymatic disorders in humans. The first hereditary human disease for which an enzymatic disorder could be shown was methemoglobinemia with a recessive mode of inheritance (Gibson and Harrison, 1947; Gibson, 1948) (25080). In this case, the damaged enzyme is NADH - dependent methemoglobin reductase. The first attempt to systematically study a group of human diseases associated with metabolic defects was made in 1951. In a study of glycogen storage disease, the Corys showed that in eight out of ten cases of the pathological condition that was diagnosed as Gierke's disease (23220), the structure of liver glycogen was a normal variant, and in two cases it was clearly disturbed. It was also evident that liver glycogen, accumulating in excess, could not be directly converted into sugar, since patients tend to hypoglycemia. Many enzymes are needed to break down glycogen into glucose in the liver. Two of them, amyl-1,6-glucosidase and glucose-6-phosphatase, were chosen for study as possible defective elements of the enzyme system. Phosphate release from glucose-6phosphate was measured in liver homogenates at various pH values. The results are presented in fig. 4.2. In a normal liver, high activity was found with an optimum at pH 6-7. Severe liver dysfunction in cirrhosis correlated with a slight decrease in activity. On the other hand, in the case of Gierke's disease with a fatal outcome, the activity of the enzyme could not be detected at all; the same result was obtained in the examination of the second similar patient. In two patients with less severe symptoms, there was a significant decrease in activity.

It was concluded that in these cases of Gierke's disease with a fatal outcome, there was a defect in glucose-6-phosphatase. However, in most of the milder cases, the activity of this enzyme was not lower than in liver cirrhosis, and only in two patients was it slightly lower (Fig. 4.2).

According to the Corey spouses, the abnormal accumulation of glycogen in muscle tissue cannot be associated with a lack of glucose-6-phosphatase, since this enzyme is absent in the muscles and is normal. As a possible explanation for muscle glycogenosis, they suggested a violation of the activity of amylo-1,6-glucosidase. This prediction was soon confirmed: Forbes discovered such a defect in one of the clinically significant cases of glycogen storage disease involving the heart and skeletal muscles. Now we

4. Action of genes 11

a large number of enzymatic defects are known in glycogen storage disease.

Although the various forms of this disease vary somewhat in degree of manifestation, there is much in common between them clinically. With one exception, they are all inherited in an autosomal recessive manner. If enzymatic defects had not been uncovered, the pathology of glycogen accumulation would be considered as a single disease with characteristic intrafamilial correlations in severity, symptom details, and timing of death. Thus, we have an example where genetic heterogeneity, which could only be assumed on the basis of the study of the phenotype (Sec. 3.3.5), was confirmed by analysis at the biochemical level: the study of enzymatic activity made it possible to identify specific genes.

In subsequent years, the pace of research into enzymatic defects increased, and for the 588 identified recessive autosomal genes that McKusick describes in the sixth edition of his book Mendelian Inheritance in Man (1983), specific enzymatic disorders were found in more than 170 cases. Our progress in this area is directly related to the development of the concepts and methods of molecular genetics.

Some stages of the study of enzymatic disorders in humans. We present only the most important milestones in this ongoing process: 1934 Völling discovered phenylketonuria

1941 Beadle and Tatum formulated the one-gene-one-enzyme hypothesis 1948 Gibson described the first case of an enzymatic disorder in a human disease (recessive methemoglobinemia)

1952 Cory's discovered glucose-6-phosphatase deficiency in Gierke's disease

1953 Jervis demonstrated the absence of phenylalanine hydroxylase in phenylketonuria. Bickel reported the first attempt to alleviate an enzymatic disorder by adopting a diet low in phenylalanine.

1955 Smithies developed the starch gel electrophoresis technique

1956 Carson et al. discovered a defect in glucose-6-phosphate dehydrogenase (G6PD) in a case of induced hemolytic anemia

1957 Kalkar et al. described enzymatic deficiency in galactosemia, showing that humans and bacteria have an identical enzymatic disorder

1961 Krut and Weinberg demonstrated an enzyme defect in galactosemia in vitro in cultured fibroblasts

1967 Sigmiller et al. discovered a hypoxanthine-guanine phosphoribosyltransferase (HPRT) defect in Lesch-Nyhan syndrome

1968 Cleaver described violation of excisional repair in xeroderma pigmentosa

1970 Neufeld identified enzymatic defects in mucopolysaccharidoses, which made it possible to identify the pathways for the breakdown of mucopolysaccharides

1974 Brown and Goldstein proved that the genetically determined overproduction of hydroxymethylglutaryl-CoA reductase in familial hypercholesterolemia is due to a defect in the membrane-located low-density lipoprotein receptor, which modulates the activity of this enzyme (HMG)

1977 Sly et al. demonstrated that mannose-6-phosphate (as a component of lysosomal enzymes) is recognized by fibroblast receptors. A genetic defect in processing prevents the binding of lysosomal enzymes, resulting in impaired release into the cytoplasm and subsequent secretion into the plasma (I-cell disease)

12 4. Action of genes

1980 In pseudohypoparathyroidism, a defect in the protein that provides the coupling of the receptor and cyclase was discovered.

Genetics- science is by no means young, research in it has been going on for several centuries, starting with Mendel in 1865 and up to the present day. The term "gene" for a unit of hereditary characteristic was first proposed by Johannsen in 1911, and in the 1940s was refined by the concept of "one gene - one enzyme" proposed by Tatum and Beadle.

This position was determined in experiments on Drosophila flies, but equally applies to humans; Ultimately, the life of all beings is determined by their DNA. The DNA molecule in humans is larger than in all other organisms, and it is more complex, but the essence of its functions is the same for all living beings.

Concept " one gene - one enzyme”, which arose on the basis of the ideas of Tatum and Beadle, can be formulated as follows:
1. All biological processes are under genetic control.
2. All biochemical processes occur in the form of phased reactions.
3. Each biochemical reaction is ultimately controlled by different individual genes.
4. A mutation in a certain gene leads to a change in the cell's ability to carry out a certain chemical reaction.

Since then, the concept of "one gene - one enzyme" has expanded somewhat, and now sounds like " one gene - one protein". In addition, recent research suggests that some genes work in conjunction with others to produce unique proteins, meaning that some genes can code for more than one protein.

human genome contains about 3 billion nucleotide pairs; it is believed that it contains from 50,000 to 100,000. After deciphering the genome, it turned out that there were only about 30,000 genes. The interaction of these genes is much more complicated than expected. Genes are encoded in DNA strands, which, in combination with certain nuclear proteins, form chromosomes.

Genes- not just segments of DNA: they are formed by coding sequences - exons, interspersed with non-coding sequences - introns. Exons, as the expressed part of DNA, are only a small part of the most important molecule of the organism; most of it is not expressed, is formed by introns and is often called "silent" DNA.

Approximate size and structure human genome shown in the figure below. The functional length of the human chromosome is expressed in centimorganides. Centimorganide (cm) - the distance over which the probability of crossing over during meiosis is 1%. Gene linkage analysis has shown that the length of the human genome is about 3000 cM.

Medium chromosome contains approximately 1500 genes, encoded in 130 million base pairs. The figure below schematically shows the physical and functional dimensions of the genome: the first one is calculated in nucleotide pairs, and the second one is in cM. Most of the human genome is represented by "silent" DNA and is not expressed.

On DNA template As a result of the transcription process, RNA is synthesized, and then protein. Therefore, the DNA sequence completely determines the sequence of the functional proteins of the cell. All proteins are synthesized as follows:
DNA => RNA => protein

The genetic apparatus of humans and other mammals is more complex than that of other living organisms, since sections of some genes in mammals can be combined with parts of others genes, resulting in the synthesis of an entirely new protein or the control of a particular cellular function.

Therefore, it is possible for a person to increase the number of genes expressed without actually increasing the amount of genes expressed. DNA or the absolute number of genes.
Overall, about 70% of all genetic material is not expressed.

The discoveries of the exon-intron organization of eukaryotic genes and the possibility of alternative splicing have shown that the same nucleotide sequence of the primary transcript can provide the synthesis of several polypeptide chains with different functions or their modified analogs. For example, yeast mitochondria contain the box (or cob) gene encoding the cytochrome b respiratory enzyme. It can exist in two forms (Fig. 3.42). The “long” gene, consisting of 6400 bp, has 6 exons with a total length of 1155 bp. and 5 introns. The short form of the gene consists of 3300 bp. and has 2 introns. It is actually a "long" gene devoid of the first three introns. Both forms of the gene are equally well expressed.

After the removal of the first intron of the “long” box gene, based on the combined nucleotide sequence of the first two exons and part of the nucleotides of the second intron, a template for an independent protein, RNA maturase, is formed (Fig. 3.43). The function of RNA maturase is to provide the next stage of splicing - the removal of the second intron from the primary transcript and, ultimately, the formation of a template for cytochrome b.

Another example is a change in the splicing pattern of the primary transcript encoding the structure of antibody molecules in lymphocytes. The membrane form of antibodies has a long "tail" of amino acids at the C-terminus, which ensures the fixation of the protein on the membrane. The secreted form of antibodies does not have such a tail, which is explained by the removal of nucleotides encoding this region from the primary transcript during splicing.

In viruses and bacteria, a situation has been described where one gene can simultaneously be part of another gene, or some DNA nucleotide sequence can be part of two different overlapping genes. For example, on the physical map of the phage FX174 genome (Fig. 3.44), it can be seen that the B gene sequence is located inside the A gene, and the E gene is part of the D gene sequence. This feature of the organization of the phage genome managed to explain the existing discrepancy between its relatively small size (it consists of 5386 nucleotides) and the number of amino acid residues in all synthesized proteins, which exceeds the theoretically permissible for a given genome capacity. The possibility of assembling different peptide chains on mRNA synthesized from overlapping genes (A and B or E and D) is ensured by the presence of ribosomal binding sites within this mRNA. This allows translation of another peptide to start from a new point of reference.

The nucleotide sequence of the B gene is also part of the A gene, and the E gene is part of the D gene.

In the λ phage genome, overlapping genes were also found, translated both with a frameshift and in the same reading frame. It is also assumed that two different mRNAs can be transcribed from both complementary strands of the same DNA region. This requires the presence of promoter regions that determine the movement of RNA polymerase in different directions along the DNA molecule.

The described situations, which testify to the admissibility of reading different information from the same DNA sequence, suggest that overlapping genes are a fairly common element in the organization of the genome of viruses and, possibly, prokaryotes. In eukaryotes, gene discontinuity also allows the synthesis of various peptides based on the same DNA sequence.

With all of this in mind, it is necessary to amend the definition of a gene. Obviously, one can no longer speak of a gene as a continuous sequence of DNA that uniquely encodes a specific protein. Apparently, at present, the formula "One gene - one polypeptide" should still be considered the most acceptable, although some authors suggest changing it: "One polypeptide - one gene." In any case, the term gene should be understood as a functional unit of hereditary material, which by its chemical nature is a polynucleotide and determines the possibility of synthesizing a polypeptide chain, tRNA or rRNA.

One gene one enzyme.

In 1940, J. Beadle and Edward Tatum used a new approach to study how genes provide metabolism in a more convenient object of research - the microscopic fungus Neurospora crassa .. They obtained mutations in which; there was no activity of one or another metabolic enzyme. And this led to the fact that the mutant fungus was not able to synthesize a certain metabolite itself (for example, the amino acid leucine) and could live only when leucine was added to the nutrient medium. The theory "one gene - one enzyme" formulated by J. Beadle and E. Tatum quickly gained wide recognition among geneticists, and they themselves were awarded the Nobel Prize.

Methods. selection of the so-called "biochemical mutations" that lead to disruption of the action of enzymes that provide different metabolic pathways, proved to be very fruitful not only for science, but also for practice. First, they led to the emergence of genetics and selection of industrial microorganisms, and then to the microbiological industry, which uses strains of microorganisms that overproduce such strategically important substances as antibiotics, vitamins, amino acids, etc. The principles of selection and genetic engineering of strains of overproducers are based on the notion that "one gene codes for one enzyme". And although this idea is excellent practice brings multi-million dollar profits and saves millions of lives (antibiotics) - it is not final. One gene is not just one enzyme.

Discoveries of the exon-intron organization of eukaryotic genes and the possibility of alternative splicing have shown that the same nucleotide sequence of the primary transcript can provide the synthesis of several polypeptide chains with different functions or their modified analogs. For example, yeast mitochondria contain the box (or cob) gene encoding the cytochrome b respiratory enzyme. It can exist in two forms: The “long” gene, consisting of 6400 bp, has 6 exons with a total length of 1155 bp. and 5 introns. The short form of the gene consists of 3300 bp. and has 2 introns. It is actually a "long" gene devoid of the first three introns. Both forms of the gene are equally well expressed.