Processing, splicing. The role of RNA in the process of realization of hereditary information
Capping and polyadenylation of mRNA is called processing ( post-transcriptional modification).
Capping:
A residue is attached to the 5" end of all eukaryotic mRNAs during processing. 7-methylguanosine with education unique 5"à 5" phosphodiester bond. This extra nucleotide is called cap or cap.
Cap functions :
1. it protects RNA from exonucleases
2. helps the binding of the mRNA molecule to the ribosome.
Polyadenylation:
The 3"-end is also modified immediately after transcription is completed. Special enzyme - polyadenylate polymerase attaches from 20 to 250 adenylic acid residues (poly (A)) to the 3 "end of each RNA transcript. Polyadenylate polymerase recognizes a specific sequence aaaaa, cleaves a small fragment of 11-30 nucleotides from the primary transcript and then adds a poly(A) sequence. It is generally accepted that such a "tail" facilitates the subsequent processing of RNA and the export of mature mRNA molecules from the nucleus.
As mRNA participates in translation processes, the length of the polyA fragment decreases. 30 adenyl nucleotides are considered critical for stability.
The entire set of nuclear transcripts of RNA polymerase II is known as heterogeneous nuclear RNA(hnRNA).
All 3 classes of RNA are transcribed from genes that contain introns(non-informative sections) and exons(sections of DNA that carry information). The sequences encoded by DNA introns must be removed from the primary transcript before the RNA becomes biologically active. The process of removing copies of intron sequences is called RNA splicing.
RNA splicing is catalyzed complexes of proteins with RNA, known as "small nuclear ribonucleoprotein particles"(snRNP, English small nuclear ribonucleic particles, snRNP). Such catalytic RNAs are called ribozymes.
Functions of introns:
protect the functionally active part of the cell genome from the damaging effects of chemical or physical (radiation) factors
allows using the so-called alternative splicing increase the genetic diversity of the genome without increasing the number of genes.
Alternative splicing:
As a result of a change in the distribution of exons of one transcript during splicing, different RNAs and, consequently, different proteins arise.
More than 40 genes are already known, the transcripts of which are subjected to alternative splicing. For example, a transcript of the calcitonin gene, as a result of alternative splicing, produces RNA that serves as a template for the synthesis of calcitonin (in the thyroid gland) or a specific protein responsible for taste perception (in the brain). The transcript of the -tropomyosin gene undergoes even more complex alternative splicing. At least 8 different tropomyosin mRNAs derived from a single transcript have been identified (see figure)
33 . The general scheme of protein biosynthesis - necessary prerequisites:
Information flow - information transfer scheme (the central dogma of molecular biology). DNA replication and transcription - enzymes, mechanism. Reverse transcription, the role of revertases. mRNA processing and splicing. Characteristics of the genetic code, codon, anticodon.
The difference between protein biosynthesis and the biosynthesis of other molecules:
There is no correspondence between the number of matrix monomers and in the reaction product (4 nucleotides - 20 amino acids)
There is no complementarity between the mRNA (template) and the peptide chain of the protein (product).
The general scheme of protein biosynthesis - necessary prerequisites:
· information flow(transfer of information from DNA to RNA to protein)
· plastic flow(amino acids, mRNA, tRNA, enzymes)
· energy flow(macroergy ATP, GTP, UTP, CTP)
It is this stage that distinguishes the implementation of the available genetic information in such cells as eukaryotes and prokaryotes.
Interpretation of this concept
Translated from English, this term means "processing, processing." Processing is the process of formation of mature ribonucleic acid molecules from pre-RNA. In other words, this is a set of reactions that lead to the transformation of primary transcription products (pre-RNA of different types) into already functioning molecules.
As for the processing of p- and tRNA, it most often comes down to cutting off excess fragments from the ends of the molecules. If we talk about mRNA, then it can be noted that in eukaryotes this process proceeds in many stages.
So, after we have already learned that processing is the transformation of a primary transcript into a mature RNA molecule, it is worth moving on to considering its features.
The main features of the concept under consideration
These include the following:
- modification of both the ends of the molecule and RNA, in the course of which specific nucleotide sequences are attached to them, showing the place of the beginning (end) of translation;
- splicing - cutting off non-informative ribonucleic acid sequences that correspond to DNA introns.
As for prokaryotes, their mRNA is not subject to processing. It has the ability to work immediately after the completion of the synthesis.
Where does the process in question take place?
In any organism, RNA processing takes place in the nucleus. It is carried out by means of special enzymes (their group) for each individual type of molecule. Translation products such as polypeptides that are directly read from mRNA can also be processed. The so-called precursor molecules of most proteins - collagen, immunoglobulins, digestive enzymes, some hormones - undergo these changes, after which their real functioning in the body begins.
We have already learned that processing is the process of forming mature RNAs from pre-RNAs. Now it is worth delving into the nature of ribonucleic acid itself.
RNA: chemical nature
It is a copolymer of pyrimidine and purine ribonucleitides, which are connected to each other, in the same way as in DNA, by 3' - 5'-phosphodiester bridges.
Despite the fact that these 2 types of molecules are similar, they differ in several ways.
Differences between RNA and DNA
Firstly, ribonucleic acid has a carbon residue, which is adjacent to pyrimidine and purine bases, phosphate groups - ribose, while DNA has 2'-deoxyribose.
Secondly, the pyrimidine components also differ. Similar components are the nucleotides of adenine, cytosine, guanine. RNA contains uracil instead of thymine.
Thirdly, RNA has a 1-stranded structure, while DNA is a 2-stranded molecule. But in the ribonucleic acid chain there are regions with opposite polarity (complementary sequence), due to which its single chain is able to fold and form "hairpins" - structures endowed with 2-helix characteristics (as shown in the figure above).
Fourthly, in view of the fact that RNA is a single strand, which is complementary only to the 1st of the DNA strands, guanine does not have to be present in it in the same content as cytosine, and adenine - like uracil.
Fifth, RNA can be hydrolyzed with alkali to 2', 3'-cyclic diesters of mononucleotides. The role of an intermediate product in hydrolysis is played by 2', 3', 5-triester, which is incapable of formation in the course of a similar process for DNA due to the absence of 2'-hydroxyl groups in it. Compared to DNA, the alkaline lability of ribonucleic acid is a useful property for both diagnostic and analytical purposes.
This sequence is complementary to the gene chain (coding) from which the RNA is “read”. Because of this property, a ribonucleic acid molecule can specifically bind to a coding strand, but is unable to do so with a non-coding DNA strand. The RNA sequence, except for the replacement of T with U, is similar to that of the non-coding strand of the gene.
RNA types
Almost all of them are involved in such a process as the following types of RNA are known:
- Matrix (mRNA). These are cytoplasmic ribonucleic acid molecules that act as templates for protein synthesis.
- Ribosomal (rRNA). This is a cytoplasmic RNA molecule that acts as structural components such as ribosomes (an organelle involved in protein synthesis).
- Transport (tRNA). These are molecules that take part in the translation (translation) of mRNA information into a sequence of amino acids already in proteins.
A significant part of RNA in the form of 1st transcripts, which are formed in mammalian cells, is subject to degradation in the nucleus and does not play an informational or structural role in the cytoplasm.
In human cells (cultivated), a class of small nuclear ribonucleic acids was found that are not directly involved in protein synthesis, but affect RNA processing, as well as the overall cellular "architecture". Their sizes vary, they contain 90 - 300 nucleotides.
Ribonucleic acid is the main genetic material in a number of plant and animal viruses. Some RNA viruses never go through the same RNA-to-DNA stage. But still, many animal viruses, for example, retroviruses, are characterized by reverse translation of their RNA genome, directed by RNA-dependent reverse transcriptase (DNA polymerase) with the formation of a 2-stranded DNA copy. In most cases, the emerging 2-stranded DNA transcript is introduced into the genome, further providing the expression of viral genes and the production of the latest copies of RNA genomes (also viral).
Post-transcriptional modifications of ribonucleic acid
Its molecules synthesized with RNA polymerases are always functionally inactive and act as precursors, namely pre-RNA. They are transformed into already mature molecules only after they have passed the corresponding post-transcriptional modifications of RNA - the stages of its maturation.
The formation of mature mRNA begins during the synthesis of RNA and polymerase II at the elongation stage. Already to the 5'-end of the gradually growing RNA strand is attached by the 5'-end of GTP, then the orthophosphate is cleaved off. Further, guanine is methylated with the appearance of 7-methyl-GTP. Such a special group, which is part of the mRNA, is called a "cap" (hat or cap).
Depending on the type of RNA (ribosomal, transport, template, etc.), precursors undergo various sequential modifications. For example, mRNA precursors undergo splicing, methylation, capping, polyadenylation, and sometimes editing.
Eukaryotes: general characteristics
The eukaryotic cell is the domain of living organisms, and it contains the nucleus. In addition to bacteria, archaea, any organisms are nuclear. Plants, fungi, animals, including the group of organisms called protists, are all eukaryotic organisms. They are both 1-celled and multicellular, but they all have a common plan of the cellular structure. It is generally accepted that these so dissimilar organisms have the same origin, which is why the nuclear group is perceived as a monophyletic taxon of the highest rank.
Based on popular hypotheses, eukaryotes arose 1.5 - 2 billion years ago. An important role in their evolution is given to symbiogenesis - the symbiosis of a eukaryotic cell that had a nucleus capable of phagocytosis and bacteria swallowed by it - the precursors of plastids and mitochondria.
Prokaryotes: general characteristics
These are 1-celled living organisms that do not have a nucleus (formed), the rest of the membrane organelles (internal). The only large circular 2-stranded DNA molecule that contains the bulk of the cellular genetic material is one that does not form a complex with histone proteins.
Prokaryotes include archaea and bacteria, including cyanobacteria. Descendants of non-nuclear cells - eukaryotic organelles - plastids, mitochondria. They are subdivided into 2 taxa within the domain rank: Archaea and Bacteria.
These cells do not have a nuclear envelope; DNA packaging occurs without the involvement of histones. The type of their nutrition is osmotrophic, and the genetic material is represented by one that is closed in a ring, and there is only 1 replicon. Prokaryotes have organelles that have a membrane structure.
The difference between eukaryotes and prokaryotes
The fundamental feature of eukaryotic cells is associated with the presence of a genetic apparatus in them, which is located in the nucleus, where it is protected by a membrane. Their DNA is linear, associated with histone proteins, other chromosomal proteins that are absent in bacteria. As a rule, they contain 2 nuclear phases. One has a haploid set of chromosomes, and subsequently merging, 2 haploid cells form a diploid cell, which already contains the 2nd set of chromosomes. It also happens that during subsequent division, the cell again becomes haploid. This kind of life cycle, as well as diploidy in general, is not characteristic of prokaryotes.
The most interesting difference is the presence of special organelles in eukaryotes, which have their own genetic apparatus and reproduce by division. These structures are surrounded by a membrane. These organelles are plastids and mitochondria. In terms of vital activity and structure, they are surprisingly similar to bacteria. This circumstance prompted scientists to think that they are the descendants of bacterial organisms that entered into symbiosis with eukaryotes.
Prokaryotes have few organelles, none of which are surrounded by a 2nd membrane. They lack the endoplasmic reticulum and lysosomes.
Another important difference between eukaryotes and prokaryotes is the presence of the phenomenon of endocytosis in eukaryotes, including phagocytosis in most groups. The latter is the ability to capture by means of confinement in a membrane bubble, and then digest various solid particles. This process provides the most important protective function in the body. The occurrence of phagocytosis is presumably due to the fact that their cells are of medium size. Prokaryotic organisms, on the other hand, are incommensurably smaller, which is why in the course of eukaryotic evolution, a need arose associated with supplying the cell with a significant amount of food. As a result, the first mobile predators arose among them.
Processing as one of the stages of protein biosynthesis
This is the second step that starts after transcription. Protein processing occurs only in eukaryotes. This is mRNA maturation. To be precise, this is the removal of regions that do not code for a protein, and the addition of controls.
Conclusion
This article describes what processing is (biology). It also tells what RNA is, lists its types and post-transcriptional modifications. The distinctive features of eukaryotes and prokaryotes are considered.
Finally, it is worth recalling that processing is the process of formation of mature RNA from pre-RNA.
| Parameter name | Meaning |
| Article subject: | RNA processing |
| Rubric (thematic category) | Biology |
Primary RNAs (RNA precursors, heterogeneous nuclear RNAs) resulting from transcription are, in most cases, functionally inactive molecules. For this reason, immediately after transcription, they undergo a series of modifications and turn into mature RNAs. The maturation of primary transcripts is called processing.
Rice. 32. ρ-
dependent transcription termination in bacteria
Processing of mRNA precursors is not characteristic of bacterial cells and is required only for the formation of mature rRNA and tRNA molecules.
RNA processing in eukaryotes is a rather complex and finely organized process that directly affects the regulation of the expression of genetic material. Eukaryotic mRNA processing has been studied in the most detail, including:
splicing - excision of non-coding regions (introns) from pre-mRNA and stitching together regions (exons) encoding the protein structure;
capping - the formation of a special structure at the 5'-end of mRNA - a cap - occurs shortly after the start of mRNA synthesis and is carried out with the participation of GTP;
polyadenylation - the formation of a poly(A) fragment at the 3'-end containing about 200 adenyl nucleotides (Fig. 33).
Rice. 33. mRNA processing
splicing mechanism
Splicing of pre-mRNA eukaryotes involves a number of proteins, as well as a special type of RNA - small nuclear RNA (snRNA). Various snRNAs bind to the border regions of RNA introns according to the principle of complementarity. For this interaction, certain sequences of nucleotides at the beginning and end of introns are essential: for example, introns always begin with G-U, and end with an A-G doublet. Small nuclear RNAs form a complex with enzymes that catalyze splicing - spliosome.
The first pre-RNA break occurs at the 5' end of the intron, which binds to one of the nucleotides in the middle part of the same intron (Fig. 34). This leads to the formation of a ring (or, more precisely, a lasso-like) structure. The first snRNA dissociates, and the enzyme complex moves to another snRNA, which marks the 3' end of the intron. This is where the second pre-RNA break occurs. The connection of exon 2 to the intron is replaced by the connection to exon 1.
Alternative splicing
In some cases, it is possible to change the course of splicing and implement it in an alternative way. In this case, more than one type of mRNA is read from one gene. Alternative splicing allows an organism to synthesize proteins of different structure and properties based on a single gene. Such genes encode families of related proteins involved in muscle contraction, the formation of the cytoskeleton of nerve 
fibers, peptide hormones, etc.
Rice. 34. Probable spicing mechanism:
E - enzyme complex (with nuclease and ligase activity)
Alternative mRNA splicing involves three basic mechanisms:
1. Use of different promoters. In the presence of alternative promoters in the gene, different types of RNA can be synthesized from different sites of transcription initiation. An alternative promoter is a complex promoter consisting of at least two independently functioning parts located before different exons of the same gene. In this case, transcripts are formed that have 5′ ends of different lengths and a different number of exons.
2. Change in the polyadenylation site of the primary transcript. As a result, the size and structure of the 3'-terminal region of pre-mRNA changes.
3. Connection of exons in various combinations. In this case, some exons may not be included in splicing. For example, if a gene contains only six exons (from 1st to 6th), in one type of mRNA they can be arranged in the order 1,2,3,4,5,6, in other RNAs the order should be different, for example 4,5,6,1,2,3, or 2,5,6, or 1,3,5.
Alternative splicing provides fine regulation of genes in eukaryotes, tissue differentiation, and determines the development of various traits determined by one gene. In humans, about 1/3 of all genes can encode more than one protein, i.e. different proteins are encoded by different combinations of exons of the same gene. The presence of alternative splicing may explain the fact that the number of proteins in the human body is several times greater than the number of protein-coding genes.
RNA processing - concept and types. Classification and features of the category "RNA Processing" 2017, 2018.
Processing in eukaryotes affects all types of primary transcripts of eukaryotic genes.
Processing in eukaryotes
capping is the formation of a special structure at the 5 "end of mRNA - a cap (cap). Capping occurs even before transcription is complete and protects the 5" end of RNA from the action of nucleases. RNA capping is carried out with the participation of GTP(guanosine triphosphate), from which GMP is transferred to the 5'-diphosphate of the first mRNA nucleotide.
Polyadenylation carried out by the enzyme poly (A) polymerase and leads to the formation at the 3 "end of the oligo (A) fragment containing 100 - 200 adenylic acid residues in a row and also called the "poly (A) tail." This poly (A) -subsequence added to RNA after cap attachment. First, the 3" end of the RNA is cleaved off by enzymes at a point 10–35 ribonucleotides away from the conservative AAUAAAA sequence, and then polyadenylation of this end of the RNA molecule occurs. The poly(A) tail is found in almost all mRNA eukaryotic organisms, with the exception of histone gene transcripts. The AAUAAAA sequence is not found in all eukaryotic RNA transcripts. Apparently, this is due to mutations that prevent polyadenylation. In the absence of a 3" tail, RNA transcripts are rapidly degraded by enzymes.
That. The 5' cap and 3' tail are extremely important for further processing and transport of mRNA into the cytoplasm. The poly(A) tail determines the stability of the mRNA and its lifetime in the cell. In addition, it promotes the release of mRNA from the nucleus into the cytoplasm, and is also essential for the regulation of translation.
Splicing mechanisms: RNA autocatalysis (Klag,400)
Different types of nuclear RNA, as well as mth and hlp RNAs, have their own splicing mechanisms.
Depending on the specificity of the splicing mechanism, introns can be divided into several groups. To the first group include introns that are part of the primary rRNA transcript, the removal of which does not require additional components. These introns themselves have the enzymatic activity necessary for their excision. This fact was first discovered in 1982 (Tomas Cech et al.) in the flagellar protozoan Tetrachymena. Because of their autocatalytic properties, self-splicing RNAs are sometimes referred to as ribozymes .
The process of self-cutting (auto-excision) (Fig. 145_Konichev)
(Fig. 12-12, Klag) is two nucleophilic reactions, or reactions transesterification, in which guanosine interacts with the primary itranscript and acts as a cofactor. In this case, the 3'-hydroxyl group of guanosine is transferred to the nucleotide adjacent to the 5'-end of the intron. In the second reaction, this hydroxyl group interacts with a phosphate group at the 3' end of the right intron, as a result, the intron is excised, and the ends of two adjacent exons are joined to form mature mRNA.
The 26S rRNA intron of tetrachymene - IVS, consists of 413 nucleotides. As a result of the reaction transesterification ligation of two exons with the formation of mature 26S rRNA is carried out without additional energy expenditure. The excised intron is then cyclized. A fragment containing 19 nucleotides is released from its composition by two-stage autocleavage, resulting in the formation of RNA 376 nucleotides long (L -19 IVS), which is a true RNA enzyme (ribozyme) with catalytic properties. This ribozyme has a stable structure, has endonuclease activity, cleaving long single-stranded RNAs, and exhibits specificity, recognizing CUCU tetranucleotides in the composition of the attacked substrate. In structure type I introns characteristic internal oligopurine sequences have been identified (in tetrahymenes this is the GGAGGG sequence), called adapter sequences , which are involved in the formation of the active center of RNA enzymes and play an important role in the catalytic cleavage of RNA.
Such self-excision of introns is characteristic of pre-rRNAs of other protozoa. This mechanism apparently also operates during the removal of introns from the primary mRNA and tRNA transcripts in mitochondria and chloroplasts, which are related to group II.
To cut introns second group two autocatalytic reactions are also needed, but guanosine is not needed.
Further studies made it possible to establish that not only large RNAs (~400 nucleotides in tetrachymenes and RNase P) but also short 13-20-mer oligonucleotides that can be synthesized in vitro have catalytic activity. These ribozymes are called miniwinters . One of the detailed models of the functioning of such ribozymes is called "hammer-head "(Fig. 146). The tertiary structure of the "hammer head" is stabilized by divalent metal ions, which neutralize the negatively charged oxygen atoms of the phosphodiester bonds and simultaneously connect the phosphate groups by covalent bonds, which is essential for the formation of a stable transition state (enzyme-substrate complex). As in the case of catalysis carried out by enzymes of a protein nature, ribozymes and the attacked substrate
(natural or synthetically obtained RNA molecules) form an enzyme-substrate complex, and then an enzyme-product complex (see Fig. 146).
Splicing mechanisms: spliceosome. (mRNA processing in eukaryotes)
In nuclear pre-mRNAs, introns can be up to 20,000 nucleotides long. Therefore, their removal requires a more complex mechanism than self-cutting (auto-excision). (Fig.12-13). The nucleotide sequences at the ends of the introns in these molecules are similar: the 5' ends often contain a dinucleotide (GU) GU, and at the 3 "-end - a dinucleotide (AG) AG. Molecules of special proteins bind to these sequences, which form a complex called splicesome. The main component of the spliceosome is small nuclear ribonucleoproteins, or snRNPs, which are found only in the nucleus and are enriched in uridine residues. Therefore, small nRNAs are often referred to as U1 , U2 ...U6.
[Konichev, p.292. In pre-mRNA splicing
in higher eukaryotes, a number of proteins are involved, as well as a special type of RNA - small nuclear RNA (snRNA). Small nuclear RNAs have sequences ranging from 65 to 1000 or more nucleotides (10S-90S), rich in uridyl nucleotides, and therefore are also called uRNAs (Ul, U2, etc.). In yeast, 25 different snRNAs have been identified, and in vertebrates, 15. In clawed frogs Xenopus laevis, a number of snRNAs (U3, U8, U14, and U22) are involved in the processing of ribosomal RNAs by binding to the border regions of spacer sequences (see Fig. 143). Small nuclear RNAs have been found not only in vertebrates and yeasts, but also in insects and archaebacteria. They are probably a very ancient group of molecules. Nucleotide sequence of all relevant uRNAs
eukaryotes coincide by more than 90%, which, in particular, applies to U1 of humans and Drosophila. The high conservatism of the uRNA structure indicates that splicing is a very ancient process that began with autosplicing (see above) and transformed into splicing involving specific ribonucleoprotein particles, snRNPs. snRNA genes are transcribed by RNA polymerase II and have different localization in the genome: some of them are discrete independent genes,
having no introns, while the genes of other snRNAs are located inside the introns of genes encoding proteins. Thus, in Xenopus U13 is encoded by three unique sequences located
in introns 5, 6, and 8 of the heat shock protein genes, and the U16 gene is located inside the intron of the L1 ribosomal protein. The latter circumstance is important, since it shows that rRNA processing and mRNA processing of ribosome proteins can be coordinated with the participation of snRNA. Besides,
It is suggested that snRNAs can serve as RNA chaperones by participating in folding RNA, i.e. helping her to accept the necessary structure in space. Small nuclear RNAs are present in the nucleus in complexes with proteins called small ribonucleoprotein particles (snRNPs). A stable component of snRNPs is the fibrillarin protein, a very structurally conservative protein with a molecular weight of 34 kDa localized in the nucleoli. The complex consisting of many snRNPs that catalyzes the splicing of nuclear pro-mRNAs is called splicingosomes .]
It is known that snRNA type U 1 contains a nucleotide sequence homologous to the 5 "end of the intron. Pairing of these sequences gives rise to a splicesome. Then snRNA of type U2, U4, U5 and U6 joins it, splicing begins. As in the case of introns of the first group, two transesterification reactions. Z"- the hydroxyl group of adenine (A) localized in the intron interacts with the 5' splicing site, cutting the RNA strand. Then several snRNPs form an intermediate complex and the second reaction begins: the free 5' end of the intron is connected to the adenine residue. As a result, a characteristic loop-like structure of the lasso type is formed, containing a remote intron. The exon ends are then ligated and the snRNA complex releases the transcript .
[ Konichev, p.294. The interaction of various snRNAs that make up the splicingosome with the spliced pre-mRNA at the 5' and 3' sites imparts a loop-like structure to the intron. In this case, the ends of the exons approach each other, which is facilitated by the formation of non-canonical (other than Watson-Crick pairs) hydrogen bonds between two guanines contained in the 5' and 3' splicing sites (see Fig. 148). The convergence of exons creates a condition for the attack of the 3'-end of the intron by the adenyl nucleotide located near the 3'-end. As a result of the rupture of the phosphodiester bond between exon 1 and the 5"-end of the intron, the latter interacts with the adenyl nucleotide and the formation of a "lasso" loop in the intron (see Fig. 148_Konichev). Following this, the freed 3"-OH-end of exon 1 cuts 3 "- splicing site, cleaves the intron and, connecting with exon 2, eventually forms a mature (spliced) mRNA molecule ]
RNA processing (RNA post-transcriptional modifications) is a set of processes in eukaryotic cells that lead to the transformation of the primary RNA transcript into mature RNA.
The most well-known is the processing of messenger RNAs, which undergo modifications during their synthesis: capping, splicing, and polyadenylation. Ribosomal RNAs, transfer RNAs, and small nuclear RNAs are also modified (by other mechanisms).
Splicing (from the English splice - to splice or glue the ends of something) is the process of cutting out certain nucleotide sequences from RNA molecules and connecting the sequences that remain in the "mature" molecule during RNA processing. Most often, this process occurs during the maturation of messenger RNA (mRNA) in eukaryotes, while by biochemical reactions involving RNA and proteins, sections that do not code for proteins (introns) are removed from mRNA and sections encoding the amino acid sequence - exons - are connected to each other. Thus, immature pre-mRNA is converted into mature mRNA, from which cell proteins are read (translated). Most protein-coding genes in prokaryotes do not have introns, so pre-mRNA splicing is rare in them. Eukaryotes, bacteria, and archaea also have splicing of transfer RNAs (tRNAs) and other non-coding RNAs.
Processing and splicing are able to combine structures that are distant from each other into one gene, so they are of great evolutionary importance. Such processes simplify speciation. Proteins have a block structure. For example, the enzyme is DNA polymerase. It is a continuous polypeptide chain. It consists of its own DNA polymerase and endonuclease, which cleaves the DNA molecule from the end. The enzyme consists of 2 domains that form 2 independent compact particles linked by a polypeptide bridge. There is an intron at the border between two enzyme genes. Once the domains were separate genes, and then they got closer.
Violations of such a gene structure leads to gene diseases. Violation of the structure of the intron is phenotypically imperceptible, a violation in the exon sequence leads to mutation (mutation of globin genes).
Protein biosynthesis is a complex multi-stage process of synthesis of a polypeptide chain from amino acid residues that occurs on the ribosomes of cells of living organisms with the participation of mRNA and tRNA molecules. Protein biosynthesis can be divided into stages of transcription, processing and translation. During transcription, the genetic information encrypted in DNA molecules is read and this information is written into mRNA molecules. During a series of successive stages of processing, some fragments that are unnecessary in subsequent stages are removed from mRNA, and nucleotide sequences are edited. After the code is transported from the nucleus to the ribosomes, the actual synthesis of protein molecules occurs by attaching individual amino acid residues to the growing polypeptide chain.
The role of an intermediary, whose function is to translate the hereditary information stored in DNA into a working form, is played by ribonucleic acids - RNA.
ribonucleic acids are represented by one polynucleotide chain, which consists of four varieties of nucleotides containing sugar, ribose, phosphate and one of the four nitrogenous bases - adenine, guanine, uracil or cytosine
Matrix, or information, RNA (mRNA, or mRNA). Transcription. In order to synthesize proteins with desired properties, an "instruction" about the order in which amino acids are included in the peptide chain comes to the site of their construction. This instruction is contained in the nucleotide sequence of matrix, or information RNA (mRNA, mRNA) synthesized at the corresponding DNA regions. The process of mRNA synthesis is called transcription.
During synthesis, as the RNA polymerase moves along the DNA molecule, the single-stranded sections of DNA it has passed through are again combined into a double helix. The mRNA formed during transcription contains an exact copy of the information recorded in the corresponding section of DNA. Three adjacent mRNA nucleotides that code for amino acids are called codons. The mRNA codon sequence codes for the sequence of amino acids in the peptide chain. The mRNA codons correspond to certain amino acids (Table 1).
Transfer RNA (tRNA). Broadcast. Transfer RNA (tRNA) plays an important role in the process of using hereditary information by the cell. Delivering the necessary amino acids to the assembly site of peptide chains, tRNA acts as a translational mediator.
It has four main parts that perform different functions. The acceptor "stalk" is formed by two complementary connected terminal parts of tRNA. It is seven base pairs. The middle of these branches - the anticodon - consists of five pairs of nucleotides and contains an anticodon in the center of its loop.The anticodon is three nucleotides complementary to the mRNA codon, which encodes the amino acid transported by this tRNA to the site of peptide synthesis.
In general, different types of tRNA are characterized by a certain constancy of the nucleotide sequence, which most often consists of 76 nucleotides. The variation in their number is mainly due to the change in the number of nucleotides in the additional loop. Complementary regions that support the tRNA structure are usually conserved. The primary structure of tRNA, determined by the sequence of nucleotides, forms the secondary structure of tRNA, which has the shape of a clover leaf. In turn, the secondary structure determines the three-dimensional tertiary structure, which is characterized by the formation of two perpendicular double helixes (Fig. 27). One of them is formed by the acceptor and TψC branches, the other by the anticodon and D branches.
At the end of one of the double helixes is the transported amino acid, at the end of the other is the anticodon. These areas are the most remote from each other. The stability of the tertiary structure of tRNA is maintained due to the appearance of additional hydrogen bonds between the bases of the polynucleotide chain located in different parts of it, but spatially close in the tertiary structure.
Different types of tRNAs have a similar tertiary structure, although with some variations.
One of the features of tRNA is the presence of unusual bases in it, which arise as a result of chemical modification after the inclusion of a normal base in the polynucleotide chain. These altered bases determine the great structural diversity of tRNAs in the general plan of their structure.
14. Ribosomal cycle of protein synthesis (initiation, elongation, termination). Post-translational transformations of proteins.
Ribosomal cycle of protein synthesis. The process of interaction between mRNA and tRNA, which ensures the translation of information from the language of nucleotides into the language of amino acids, is carried out on ribosomes. The latter are complex complexes of rRNA and various proteins, in which the former form a scaffold. Ribosomal RNAs are not only a structural component of ribosomes, but also ensure their binding to a specific mRNA nucleotide sequence. This sets the start and reading frame for the formation of the peptide chain. In addition, they provide interaction between the ribosome and tRNA. Numerous proteins that make up ribosomes, along with rRNA, perform both structural and enzymatic roles.
The ribosomes of pro- and eukaryotes are very similar in structure and function. They consist of two subparticles: large and small. In eukaryotes, the small subunit is formed by one rRNA molecule and 33 different protein molecules. The large subunit combines three rRNA molecules and about 40 proteins. Prokaryotic ribosomes and mitochondrial and plastid ribosomes contain fewer components.
Ribosomes have two grooves. One of them holds the growing polypeptide chain, the other - mRNA. In addition, two tRNA-binding sites are isolated in ribosomes. Aminoacyl-tRNA is located in the aminoacyl, A-site, carrying a specific amino acid. In the peptidyl, P-section, tRNA is usually located, which is loaded with a chain of amino acids connected by peptide bonds. The formation of A- and P-sites is provided by both subunits of the ribosome.
At each moment, the ribosome shields a segment of mRNA with a length of about 30 nucleotides. This ensures the interaction of only two tRNAs with two adjacent mRNA codons (Fig. 3.31).
The translation of information into the "language" of amino acids is expressed in the gradual build-up of the peptide chain in accordance with the instructions contained in the mRNA. This process takes place on ribosomes, which provide the sequence for deciphering information using tRNA. Three phases can be distinguished during translation: initiation, elongation, and termination of peptide chain synthesis.
The initiation phase, or the beginning of peptide synthesis, consists in combining two ribosome subparticles that were previously separated in the cytoplasm at a certain mRNA site and attaching the first aminoacyl-tRNA to it. This also sets the frame for reading information contained in mRNA (Fig. 3.32).
In the molecule of any mRNA, near its 5 "-end, there is a site that is complementary to the rRNA of the small subunit of the ribosome and specifically recognized by it. Next to it is the initiating start codon AUT, which encodes the amino acid methionine. The small subunit of the ribosome connects to the mRNA in such a way that the start codon AUT is located in the region corresponding to the P-site.At the same time, only the initiating tRNA carrying methionine is able to take a place in the unfinished P-section of the small subunit and complementary connect to the start codon.After the described event, the large and small subunits of the ribosome combine to form its peptidyl and aminoacyl plots (Fig. 3.32).
By the end of the initiation phase, the P-site is occupied by aminoacyl-tRNA associated with methionine, while the A-site of the ribosome is located next to the start codon.
The described processes of translation initiation are catalyzed by special proteins - initiation factors, which are movably associated with a small subunit of the ribosome. Upon completion of the initiation phase and the formation of the ribosome-mRNA-initiating aminoacyl-tRNA complex, these factors are separated from the ribosome.
The elongation phase, or peptide elongation, includes all reactions from the formation of the first peptide bond to the attachment of the last amino acid. It is a cyclically recurring event in which there is a specific recognition of the next codon aminoacyl-tRNA located in the A-site, a complementary interaction between the anticodon and codon.
Due to the peculiarities of the three-dimensional organization of tRNA. (see section 3.4.3.1) when its anticodon is connected to an mRNA codon. the amino acid transported by it is located in the A-site, in the vicinity of the previously included amino acid located in the P-site. A peptide bond is formed between two amino acids, catalyzed by special proteins that make up the ribosome. As a result, the previous amino acid loses its connection with its tRNA and joins the aminoacyl-tRNA located in the A-site. The tRNA located at this moment in the P-site is released and goes into the cytoplasm (Fig. 3.33).
The movement of tRNA loaded with a peptide chain from the A site to the P site is accompanied by the advancement of the ribosome along the mRNA by a step corresponding to one codon. Now the next codon comes into contact with the A site, where it will be specifically "recognized" by the corresponding aminoacyl-tRNA, which will place its amino acid there. This sequence of events is repeated until the A-site of the ribosome receives a terminating codon for which no corresponding tRNA exists.
The assembly of the peptide chain is carried out at a fairly high rate, depending on temperature. In bacteria at 37 °C, it is expressed as the addition of 12 to 17 amino acids per 1 s to the subdipeptide. In eukaryotic cells, this rate is lower and is expressed as the addition of two amino acids in 1 s.
The termination phase, or the completion of polypeptide synthesis, is associated with the recognition by a specific ribosomal protein of one of the termination codons (UAA, UAG, or UGA) when it enters the A-site zone of the ribosome. In this case, water is attached to the last amino acid in the peptide chain, and its carboxyl end is separated from the tRNA. As a result, the completed peptide chain loses its connection with the ribosome, which breaks up into two subparticles (Fig. 3.34).
Post-translational transformations of proteins. The peptide chains synthesized during translation, on the basis of their primary structure, acquire a secondary and tertiary, and many-quaternary organization formed by several peptide chains. Depending on the functions performed by proteins, their amino acid sequences can undergo various transformations, forming functionally active protein molecules.
Many membrane proteins are synthesized as pre-proteins that have a leader sequence at the N-terminus that provides him with membrane recognition. This sequence is cleaved off during maturation and incorporation of the protein into the membrane. Secretory proteins also have a leader sequence at the N-terminus that ensures their transport across the membrane.
Some proteins immediately after translation carry additional amino acid pro-sequences that determine the stability of active protein precursors. During protein maturation, they are removed, allowing the transition of the inactive proprotein to the active protein. For example, insulin is first synthesized as pre-proinsulin. During secretion, the pre-sequence is cleaved off, and then proinsulin undergoes a modification in which part of the chain is removed from it and it turns into mature insulin.
I - RNA polymerase binds to DNA and begins to synthesize mRNA in the direction 5 "→ 3";
II - as the RNA polymerase advances, ribosomes are attached to the 5' end of the mRNA, starting protein synthesis;
III - a group of ribosomes follows the RNA polymerase, its degradation begins at the 5' end of the mRNA;
IV - the degradation process is slower than transcription and translation;
V - after the end of transcription, mRNA is released from DNA, translation and degradation at the 5 "end continue on it
Forming a tertiary and quaternary organization in the course of post-translational transformations, proteins acquire the ability to actively function, being included in certain cellular structures and performing enzymatic and other functions.
The considered features of the implementation of genetic information in pro- and eukaryotic cells reveal the fundamental similarity of these processes. Consequently, the mechanism of gene expression associated with transcription and subsequent translation of information that is encrypted with the help of a biological code developed as a whole even before these two types of cellular organization were formed. The divergent evolution of the genomes of pro- and eukaryotes led to differences in the organization of their hereditary material, which could not but affect the mechanisms of its expression.
The constant improvement of our knowledge about the organization and functioning of the material of heredity and variability determines the evolution of ideas about the gene as a functional unit of this material.
The relationship between a gene and a trait. Example. The hypothesis "one gene - one enzyme", its modern interpretation.
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.
