Processing - this is the maturation of preRNA synthesized on DNA and its conversion into mature RNA. Takes place in the cell nucleus in eukaryotes.
Components of processing
- Removal nucleotides. Result: a significant decrease in the length and mass of the original RNA.
- Accession nucleotides. Result: a slight increase in the length and mass of the original RNA.
- Modification(modification) of nucleotides. Result: the appearance of rare “exotic” minor (“smaller”) nucleotides in the RNA.
Nucleotide removal
1. Splitting off individual nucleotides, one at a time, from the ends of the RNA chain. Carried out by enzymes exonucleases. Typically, preRNA begins at the 5" end of ATP or GTP, and ends at the 3" end with GC regions. They are needed only for transcription itself, but are not needed for RNA to function, so they are split off.
2. Cutting off RNA fragments consisting of several nucleoids. Carried out by enzymes endonucleases. In this way, spacer nucleotide sequences are removed from the ends of preRNA.
3. Cutting preRNA into individual individual RNA molecules. Carried out by endonuclease enzymes. In this way, ribosomal RNA (rRNA) and histone RNA (mRNA) are obtained.
4. Splicing . This cutting middle sections (intronic sequences) from preRNA and then its stitching . Excision is carried out by endonuclease enzymes, and cross-linking is carried out by ligases. The result is mRNA consisting only of exonic nucleotide sequences. All pre-mRNAs are spliced, except histone ones.
As a result of the removal of nucleotides in the mRNA, for example, instead of 9200 nucleotides, only 1200 may remain.
On average, after processing, only 13% of the pre-mRNA length remains in the mature mRNA, and 87% is lost.
Addition of nucleotides
A modified 7-methylguanyl nucleotide is attached to the pre-mRNA from the initial 5" end using an atypical pyrophosphate bond; this is a component "cap" ("caps") mRNA. This cap is created at the initial stage of RNA synthesis in order to protect the nascent RNA from attacks by exonuclease enzymes that cleave off the terminal nucleotides from RNA.
After completion of the synthesis of pre-mRNA, adenyl nucleotides are sequentially added to its final section from the 3" end by the enzyme polyadenylate polymerase, so that a polyadenylate "tail" of approximately 200-250 A-nucleotides. The targets for this process are the sequences AAAAAAA and GGUUGUUGGUU at the end of the preRNA. As a result, the preRNA's own tail is cut off and replaced with a polyA tail.
Video:Supply of preRNA with a cap and tail
At the pre- tRNA tail at its 3" end is created by the sequential addition of three nucleotides: C, C and A. They form the acceptor branch of the transfer RNA.
Nucleotide modification
It is important to note that modified minor nucleotides appear in the maturing RNA as a result of processing, and are not integrated into the RNA during its synthesis on DNA.
In the nucleotides of the cap there are mRNA Ribose methylation occurs.
In pre- rRNA Ribose residues are methylated selectively along the entire length of the chain, with a frequency of approximately 1%, i.e. 1 nucleotide out of 100.
In pre- tRNA modification occurs in the most varied ways. For example, if uridine is reduced, it becomes dihydrouridine, if it is isomerized, it becomes pseudouridine, if it is methylated, it becomes methyluridine. Adenosine can be deaminated, turning into inosine, and if it is then methylated, it becomes methylinosine. Other nucleotide modifications also occur.
Video:Details about processing
Processing result
The original preRNAs are shortened and modified . Cells appear in the nucleus mature RNA different types: rRNA (28S, 18S, 5.8S, 5S), tRNA (1-3 types for each of 20 amino acids), mRNA (thousands of options depending on the number of genes expressed in a given cell). Here in the nucleus, rRNA binds to ribosomal proteins and forms large and small ribosomal subunits. They leave the nucleus and enter the cytoplasm. And the mRNA binds to transport proteins and in this form exits the nucleus into the cytoplasm.
It is this stage that distinguishes the implementation of existing genetic information in cells such as eukaryotes and prokaryotes.
Interpretation of this concept
Translated from English, this term means “processing, processing.” Processing is the process of producing 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 various types) into already functioning molecules.
As for the processing of r- and tRNA, it most often comes down to cutting off unnecessary fragments from the ends of the molecules. If we talk about mRNA, then it can be noted that in eukaryotes this process occurs in a multi-stage manner.
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 consider its features.
Main features of the concept under consideration
These include the following:
- modification of both the ends of the molecule and RNA, during which specific nucleotide sequences are attached to them, indicating the place of the beginning (end) of translation;
- splicing is the cutting off of uninformative 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 synthesis is completed.
Where does the process in question take place?
In any organism, RNA processing occurs in the nucleus. It is carried out through special enzymes (a group of them) 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 actual functioning in the body begins.
We have already learned that processing is the process of formation of mature RNA from pre-RNA. Now it’s worth delving into the nature of ribonucleic acid itself.
RNA: chemical nature
It is a copolymer of pyrimidine and purine ribonucleotides, which are connected to each other, just like in DNA, by 3' - 5' phosphodiester bridges.
Although these two types of molecules are similar, they differ in several ways.
Distinctive features of 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 are also different. Similar components are the nucleotides of adenine, cytosine, and guanine. RNA contains uracil instead of thymine.
Thirdly, RNA has a 1-stranded structure, and DNA is a 2-stranded molecule. But in the ribonucleic acid chain there are regions with opposite polarity (complementary sequence), thanks to which its single chain is able to fold and form “hairpins” - structures endowed with 2-helical characteristics (as shown in the figure above).
Fourthly, due to the fact that RNA is a single chain that is complementary to only one of the DNA chains, guanine does not necessarily 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 the intermediate product in hydrolysis is played by 2', 3', 5-triester, which is incapable of formation for DNA during a similar process due to the absence of 2'-hydroxyl groups. 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, the ribonucleic acid molecule can specifically bind to the coding strand, but is unable to do this with the 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.
Types of RNA
Almost all of them are involved in such a process as the following types of RNA are known:
- Template (mRNA). These are cytoplasmic ribonucleic acid molecules that serve as templates for protein synthesis.
- Ribosomal (rRNA). This is a cytoplasmic RNA molecule that plays the role of such structural components as ribosomes (organelles 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 first transcripts, which are formed including 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 (cultured) a class of small nuclear ribonucleic acids has been found that are not directly involved in protein synthesis, but have an impact on RNA processing, as well as the general cellular “architecture”. Their sizes vary, they contain 90 - 300 nucleotides.
Ribonucleic acid is the main genetic material of a number of plant and animal viruses. Some RNA viruses never go through the 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 incorporated into the genome, subsequently ensuring the expression of viral genes and the production of new 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 undergo the corresponding post-transcriptional modifications of RNA - the stages of its maturation.
The formation of mature mRNAs begins during RNA synthesis and polymerase II at the elongation stage. Already at the 5'-end of the gradually growing RNA strand, GTP is attached by the 5'-end, then the orthophosphate is cleaved off. Guanine is then methylated to form 7-methyl-GTP. This special group found in the mRNA is called a “cap” (cap).
Depending on the type of RNA (ribosomal, transport, template, etc.), the precursors undergo various sequential modifications. For example, mRNA precursors undergo splicing, methylation, capping, polyadenylation, and sometimes editing.
Eukaryotes: general characteristics
The eukaryotic cell acts as a domain of living organisms, and it contains the nucleus. Except bacteria, archaea, any organisms are nuclear. Plants, fungi, animals, including a group of organisms called protists, are all eukaryotic organisms. They can be both single-celled and multicellular, but they all have a common plan of cellular structure. It is generally accepted that these very different organisms have the same origin, which is why the nuclear group is perceived as a monophyletic taxon of the highest rank.
Based on common 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-cell living organisms that do not have a nucleus (formed) and other membrane organelles (internal). The only large circular 2-stranded DNA molecule containing the bulk of the genetic material of a cell is one that does not form a complex with histone proteins.
Prokaryotes include archaea and bacteria, including cyanobacteria. The descendants of nuclear-free cells are eukaryotic organelles - plastids, mitochondria. They are divided into 2 taxa within domain rank: Archaea and Bacteria.
These cells do not have a nuclear membrane; DNA packaging occurs without the involvement of histones. Their feeding type is osmotrophic, and the genetic material is represented by one which is closed in a ring, and there is only 1 replicon. Prokaryotes still have organelles that have a membrane structure.
Difference between eukaryotes and prokaryotes
The fundamental feature of eukaryotic cells is associated with the presence of the 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 one, which already contains a 2nd set of chromosomes. It also happens that with subsequent division the cell again becomes haploid. This kind of life cycle, as well as diploidity in general, is not typical for 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 their life activity and structure, they are surprisingly similar to bacteria. This circumstance prompted scientists to think that they are descendants of bacterial organisms that entered into symbiosis with eukaryotes.
Prokaryotes have a small number of organelles, none of which are surrounded by a second membrane. They lack 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 enclosing in a membrane vesicle, 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 are disproportionately smaller, which is why during the evolution of eukaryotes there arose a need 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 stage, which begins 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 (biology) is. It also explains what RNA is, its types and post-transcriptional modifications are listed. 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.
RNA processing (post-transcriptional modifications of RNA) is a set of processes in eukaryotic cells that lead to the conversion of the primary RNA transcript into mature RNA.
The best 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 joining sequences that remain in the “mature” molecule during RNA processing. This process most often occurs during the maturation of messenger RNA (mRNA) in eukaryotes, during which, through biochemical reactions involving RNA and proteins, sections of the mRNA that do not code for a protein (introns) are removed and sections that encode 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 prokaryotic protein-coding genes do not have introns, so pre-mRNA splicing is rare in them. Splicing of transfer RNAs (tRNAs) and other non-coding RNAs also occurs in representatives of eukaryotes, bacteria and archaea.
Processing and splicing are capable of combining structures that are distant from each other into a single 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 an endonuclease, which cleaves the DNA molecule from the end. The enzyme consists of 2 domains, which form 2 independent compact particles connected by a polypeptide bridge. At the border between the 2 enzyme genes there is an intron. The domains were once separate genes, but then they became closer.
Violations of such gene structure lead to gene diseases. Violation of the structure of the intron is phenotypically invisible; 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, occurring on the ribosomes of living organism cells with the participation of mRNA and tRNA molecules. Protein biosynthesis can be divided into the stages of transcription, processing and translation. During transcription, genetic information encrypted in DNA molecules is read and this information is written into mRNA molecules. During a series of successive processing stages, some fragments that are unnecessary in subsequent stages are removed from the mRNA, and nucleotide sequences are edited. After transporting the code 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 types of nucleotides containing sugar, ribose, phosphate and one of four nitrogenous bases - adenine, guanine, uracil or cytosine
Matrix, or information, RNA (mRNA, or mRNA). Transcription. In order to synthesize proteins with specified properties, “instructions” are sent to the site of their construction about the order of inclusion of amino acids in the peptide chain. This instruction is contained in the nucleotide sequence of matrix, or messenger RNA (mRNA, mRNA), synthesized in the corresponding sections of DNA. The process of mRNA synthesis is called transcription.
During the synthesis process, as RNA polymerase moves along the DNA molecule, the single-stranded DNA sections it has traversed are again combined into a double helix. The mRNA produced during transcription contains an exact copy of the information recorded in the corresponding section of DNA. Triples of adjacent mRNA nucleotides that encode amino acids are called codons. The codon sequence of the mRNA encodes the sequence of amino acids in the peptide chain. The codons of the mRNA 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 a cell. By delivering the necessary amino acids to the site of assembly of peptide chains, tRNA acts as a translational intermediary.
It has four main parts that perform different functions. The acceptor “stem” is formed by two complementary connected terminal parts of tRNA. It consists of seven base pairs. The 3" end of this stem is slightly longer and forms a single-stranded region that ends with a CCA sequence with a free OH group. The transported amino acid is attached to this end. The remaining three branches are complementary paired nucleotide sequences that end unpaired sections forming loops. The middle of these branches - the anticodon - consists of five pairs of nucleotides and contains an anticodon in the center of its loop - these are 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 changes in the number of nucleotides in the additional loop. The complementary regions that support the tRNA structure are usually conserved. The primary structure of the tRNA, determined by the nucleotide sequence, forms the secondary structure of the tRNA, which is shaped like a clover leaf. In turn, the secondary structure determines the three-dimensional tertiary structure, which is characterized by the formation of two perpendicularly located double helices (Fig. 27). One of them is formed by the acceptor and TψC branches, the other by the anticodon and D branches.
The transported amino acid is located at the end of one of the double helices, and the anticodon is located at the end of the other. These areas are located as far as possible from each other. The stability of the tertiary structure of tRNA is maintained due to the occurrence 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 tRNA have similar tertiary structures, 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 to 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 framework. Ribosomal RNAs are not only a structural component of ribosomes, but also ensure their binding to a specific nucleotide sequence of mRNA. This establishes the start and reading frame for the formation of the peptide chain. In addition, they ensure the interaction between the ribosome and tRNA. Numerous proteins that make up ribosomes, along with rRNA, perform both structural and enzymatic roles.
Ribosomes of pro- and eukaryotes are very similar in structure and function. They consist of two subparticles: large and small. In eukaryotes, the small subparticle is formed by one rRNA molecule and 33 molecules of different proteins. The large subunit combines three rRNA molecules and about 40 proteins. Prokaryotic ribosomes and ribosomes of mitochondria and plastids contain fewer components.
Ribosomes have two grooves. One of them holds the growing polypeptide chain, the other holds the mRNA. In addition, ribosomes have two tRNA binding sites. The aminoacyl A site contains an aminoacyl-tRNA carrying a specific amino acid. The peptidyl P-site usually contains tRNA, which is loaded with a chain of amino acids connected by peptide bonds. The formation of A- and P-sites is ensured by both subparticles of the ribosome.
At any given moment, the ribosome screens a segment of mRNA that is about 30 nucleotides long. This ensures the interaction of only two tRNAs with two adjacent mRNA codons (Fig. 3.31).
Translation of information into the “language” of amino acids is expressed in the gradual growth of the peptide chain in accordance with the instructions contained in the mRNA. This process occurs on ribosomes, which provide the sequence of decoding information using tRNA. During translation, three phases can be distinguished: initiation, elongation and termination of peptide chain synthesis.
The initiation phase, or the beginning of peptide synthesis, consists of the union of two ribosomal subparticles that were previously separated in the cytoplasm at a certain section of the mRNA and the attachment of the first aminoacyl-tRNA to it. This also sets the reading frame for the information contained in the mRNA (Fig. 3.32).
In the molecule of any mRNA, near its 5" end, there is a region that is complementary to the rRNA of the small ribosomal subunit and is specifically recognized by it. Next to it is located the initiating start codon OUT, which encodes the amino acid methionine. The small subunit of the ribosome connects to the mRNA in such a way that the start codon OUT is located in the region corresponding to the P-site. In this case, only the initiating tRNA carrying methionine is able to take a place in the unfinished P-site of the small subunit and complementarily combine with the start codon. After the described event, the large and small subunits of the ribosome are united with the formation of its peptidyl and aminoacyl. plots (Fig. 3.32).
By the end of the initiation phase, the P-site is occupied by aminoacyl-tRNA bound to 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 flexibly associated with the small subunit of the ribosome. Upon completion of the initiation phase and formation of the ribosome - mRNA - initiating aminoacyl-tRNA complex, these factors are separated from the ribosome.
The elongation phase, or lengthening of the peptide, includes all reactions from the moment of formation of the first peptide bond to the addition of the last amino acid. It represents cyclically repeating events in which specific recognition of the aminoacyl-tRNA of the next codon located in the A-site occurs, and a complementary interaction between the anticodon and the codon occurs.
Due to the peculiarities of the three-dimensional organization of tRNA. (see section 3.4.3.1) when connecting its anticodon to an mRNA codon. the amino acid it transports is located in the A-site, close to 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 in the P-section at this moment 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 a terminator codon, for which there is no corresponding tRNA, arrives at the A site of the ribosome.
The assembly of the peptide chain occurs at a fairly high speed, depending on temperature. In bacteria at 37 °C it is expressed in the addition of 12 to 17 amino acids per 1 s to the subpeptide. In eukaryotic cells, this rate is lower and is expressed in the addition of two amino acids per 1 s.
The termination phase, or 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 added 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 down into two subparticles (Fig. 3.34).
Post-translational transformations of proteins. Peptide chains synthesized during translation, based on their primary structure, acquire a secondary and tertiary, and many and 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 allows them to recognize the membrane. This sequence is cleaved off during maturation and insertion of the protein into the membrane. Secretory proteins also have a leader sequence at the N-terminus, which ensures their transport across the membrane.
Some proteins immediately after translation carry additional amino acid pro-sequences that determine the stability of the precursors of active proteins. When the protein matures, they are removed, ensuring the transition of the inactive protein into an 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 is converted into mature insulin.
I - RNA polymerase binds to DNA and begins to synthesize mRNA in the 5" → 3" direction;
II - as RNA polymerase advances, ribosomes are attached to the 5" end of the mRNA, beginning 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, the mRNA is freed from DNA, translation and degradation continue on it at the 5" end
By forming tertiary and quaternary organizations during post-translational transformations, proteins acquire the ability to actively function, being incorporated into 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 the transcription and subsequent translation of information, which is encrypted using the 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.
Relationship between gene and trait. Example. The “one gene - one enzyme” hypothesis, its modern interpretation.
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 analogues. For example, yeast mitochondria contain a box (or cob) gene that encodes the respiratory enzyme cytochrome b. 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 lacking the first three introns. Both forms of the gene are equally well expressed.
After removing 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 matrix is formed for an independent protein - RNA maturase (Fig. 3.43). The function of RNA maturase is to ensure the next step 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 the 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 a certain DNA nucleotide sequence can be part of two different overlapping genes. For example, the physical map of the genome of phage FX174 (Fig. 3.44) shows that the sequence of gene B is located inside gene A, and gene E is part of the sequence of gene D. This feature of the organization of the phage genome was able 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 what is 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 ribosome binding sites within this mRNA. This allows translation of another peptide to begin from a new starting point.
The nucleotide sequence of gene B is simultaneously part of gene A, and gene E is part of gene D
Overlapping genes, translated both with a frameshift and in the same reading frame, were also found in the λ phage genome. It is also assumed that it is possible to transcribe two different mRNAs from both complementary strands of one DNA section. This requires the presence of promoter regions that determine the movement of RNA polymerase in different directions along the DNA molecule.
The described situations, indicating the permissibility of reading different information from the same DNA sequence, suggest that overlapping genes are a fairly common element of the organization of the genome of viruses and, possibly, prokaryotes. In eukaryotes, gene discontinuity also allows for the synthesis of a variety of peptides from the same DNA sequence.
With all this in mind, it is necessary to amend the definition of the gene. Obviously, we can no longer talk about 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 propose to change it: “One polypeptide - one gene”. In any case, the term gene must 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 research subject - 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 on its own (for example, the amino acid leucine) and could only live when leucine was added to the nutrient medium. The “one gene, one enzyme” theory formulated by J. Beadle and E. Tatum quickly gained wide recognition among geneticists, and they themselves were awarded the Nobel Prize.
Methods. selection of so-called “biochemical mutations” leading to disturbances in the action of enzymes that provide different metabolic pathways turned out 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 superproducer strains are based on the idea that “one gene codes for one enzyme.” And although this idea is excellent for practice, brings in multimillion-dollar profits and saves millions of lives (antibiotics) - it is not final. One gene is not just one enzyme.
RNA synthesis (RNA transcription).
Structure of RNA.
Organization of genetic material in eukaryotes.
Method for recording genetic information
Organization of genetic material. Functional parts of the genome.
General information about gene expression.
1. General information about gene expression
As you know, DNA contains certain genetic information:
About the structure of all proteins and RNA of the body, as well as about the order of implementation of this information in different cells during ontogenesis and under various functional states.
Since all somatic cells of the body have the same set of 46 chromosomes, then, despite the strong differences between cells, they all contain the same genetic information in their DNA. (Some exceptions are lymphocytes, during the formation of which the rearrangement of immunoglobulin genes occurs.)
During DNA replication, genetic information is reproduced in its entirety and then passed on to daughter cells. But, in addition, this information is expressed (implemented) in the cell, determining all manifestations of its life activity. However, not all of the genetic information in the nucleus is expressed, but only some of it.
Expression of information about the structure of a particular protein includes 2 main stages:
a) The first of them is transcription: the formation in the cell nucleus on the corresponding gene (localized in one of the chromosomes) of a special messenger - messenger RNA (mRNA).
The meaning of this process is the rewriting of information about the structure of the protein from a huge stationary carrier (DNA as part of a chromosome) onto a small mobile carrier - mRNA. The situation is approximately the same when one of them is copied onto a floppy disk from a computer hard drive containing thousands of files. Therefore, mRNA read from different genes must differ from each other, just as the genes themselves differ from each other. Another important circumstance: the direct product of gene transcription is more correctly called the precursor of mRNA (pre-mRNA). The fact is that the newly formed mRNA undergoes maturation, or processing, immediately (in the nucleus). At the same time, it undergoes significant modification. And only after that the mature mRNA enters the cytoplasm from the nucleus.
b) The second of the main stages of gene expression is translation: protein synthesis on ribosomes according to a program dictated by mRNA. The essence of this program is to determine the order in which amino acids should be included in the peptide chain being built. Moreover, the process involves not free, but activated amino acids: each of them is associated with the so-called. transfer RNA (tRNA), i.e., is in the form of aminoacyl-tRNA (aa-tRNA). For each of the 20 amino acids there is its own specific form of tRNA, and more often not even one, but several forms.
Ribosomes play the role of molecular machines in translation, ensuring the correct interaction of participants. The ribosome contains four molecules, i.e. ribosomal RNA (rRNA) - one molecule of each of the 4 types of rRNA. By combining with ribosomal proteins, they form two subunits of the ribosome and perform structural and, possibly, catalytic functions in them. Thus, three classes of RNA are involved in translation - mRNA, tRNA and rRNA.
2. Organization of genetic material. Functional parts of the genome
Genes and their structure
The actual information about the structure of proteins and RNA is recorded in sections of DNA called genes and cistrons.
Gene is a section of DNA that codes for one protein.
Cistron the same section of DNA that encodes one polypeptide chain.
In animals and humans, cistrons are often located on different chromosomes and are usually also called genes. In addition to the genes for all proteins in the body, chromosomes also contain RNA genes - four types of ribosomal RNA and several dozen transfer RNAs.
The total set of genes that determine the hereditary information of an organism is called genome.
Almost all eukaryotic genes (unlike prokaryotic genes) have a characteristic feature: they contain not only coding regions - exons, but also non-coding - introns. Exons and introns are interspersed with each other, which gives the gene a “broken” structure.
The number of introns in a gene varies from 2 to several dozen; there are about 50 of them in the myosin gene. Sometimes introns account for up to 90% of the total length of the gene.
Other parts of DNA
Between genes there are also non-coding sequences - spacers. Despite the common name, their functional role can be completely different.
a) Many spacer regions apparently play a structural role:
Participate in the correct placement of the nucleosomal chain into higher chromatin structures,
In the attachment of chromosomes to the centriole apparatus, etc.
b) Other non-coding regions of DNA serve as specific binding loci for certain proteins:
Enzymes functioning on DNA
Proteins that perform a regulatory function.
In this case, the binding sites for RNA polymerase (an enzyme that synthesizes RNA on DNA) are called promoters. They are either closely adjacent to the beginning of a gene (or group of genes), or are separated from the gene by some other functional loci.
c) In eukaryotes (including humans), the regulation of the “reading” of genes is carried out not only by repressor proteins, but also by activator proteins - the so-called. transcription factors.
The latter include the already mentioned general transcription factors necessary for binding RNA polymerase to the promoter. These factors are present in all cells and are necessary to “read” any functioning gene.
Other transcription factors increase the activity of only certain genes, and the DNA loci that bind such factors are called enhancers.
d) Finally, DNA may contain short loci that serve as termination signals ( termination) DNA transcription.
Termination regions located after genes are called terminators.
3. Method of recording genetic information
Functional role of DNA strands
The two DNA strands in the gene region are fundamentally different in their functional role: one of them is coding or semantic, second - matrix.
This means that in the process of “reading” a gene (transcription, or pre-mRNA synthesis), only one DNA strand, the template, acts as a template. The product of this process, pre-mRNA, is identical in nucleotide sequence to the coding strand of DNA (with the replacement of thymine bases with uracil bases).
Thus, it turns out that with the help of the DNA template, the genetic information of the DNA coding strand is reproduced in the RNA structure during transcription.
In drawings, it is customary to depict a gene so that the coding strand is on top; then, in accordance with the general rule for depicting DNA, the 5" end of the coding strand should be located on the left.
Information on the coding circuit is written in the 5´→3´ direction; therefore, the promoter is located at the 5" end of the coding strand of the gene. And this same end is considered to be the 5" end of the entire gene (although its template strand has a 3' end here).
Basic properties of the genetic code
The unit of information in the DNA coding strand is triplet- a sequence of three nucleotides.
The 4 types of nucleotides (found in DNA) can form 64 types of triplets. Of these, 61 triplets are semantic, i.e., they encode one or another of the 20 amino acids, and 3 triplets are “senseless.”
As you can see, there are, on average, several semantic triplets per amino acid (in reality, from 1 to 6). For this reason, the genetic code is called degenerate. If it weren’t for this, random point mutations (replacements of some nucleotides in DNA with others) with a very high frequency would lead to the appearance of “meaningless” triplets.
At the same time the code specific: each of the sense triplets corresponds to only one amino acid.
The information about the protein itself is that in the complete gene (excluding introns) a linear sequence of triplets encodes a similar linear sequence of amino acids in the primary structure of this protein (in the direction from the amino to the carboxyl end of the peptide chain).
This turns out to be quite sufficient, since the primary structure of the protein determines the spatial configuration of the protein molecule, as well as its physicochemical and biological properties.
The linear correspondence between the sequence of triplets in the exons of a gene and the amino acids in the peptide chain is denoted as collinearity genetic code.
So, the genetic code is triplet. specific, degenerate, collinear and continuous. To this list is usually added versatility: in all types of organisms the meaning of any triplet is the same.
Genetic code
When we talk about code, so far we have been referring to the semantic strand of DNA. But the same, taking into account the replacement of thymine (T) with uracil (U), is the sequence of nucleotides in pre-mRNA.
Triplets of mRNA corresponding to triplets of DNA are called codons. Indeed, they are the ones directly:
The order of inclusion of amino acids in the peptide chain synthesized on the ribosome is determined.
The codons of one amino acid differ only in the last (third) nucleotide.
In amino acids that are similar in structure, the codons are also similar to each other: they match two nucleotides or one, but central, nucleotide.
4. ORGANIZATION OF GENETIC MATERIAL IN EUKARYOTES
Genes for a number of proteins and RNA
One of the distinctive features of many eukaryotic genes is the presence in their composition of non-coding regions - introns.
Another feature is that, along with unique genes (represented in a haploid genome in a single number of copies), there are repeatedly repeated genes.
To illustrate these two features, let's look at some specific genes:
Histone genes
Histones- basic (in terms of acid-base properties) proteins involved in the formation of the nucleosome structure of chromatin. Each of the five types of these proteins (HI, H2A, H2B, H3 and H4) is encoded by a corresponding gene.
Ribosomal RNA genes
Ribosomes contain four types of rRNA. These RNAs differ in sedimentation constant.
The functioning of genes is influenced by many proteins.
General transcription factors
General transcription factors are transcription factors that are necessary for the binding of RNA polymerase to the promoter, and they themselves also interact with the promoter.
p53 protein as a transcription factor
Among the large number of already discovered transcription factors, the p53 protein is perhaps the best known. This is explained by the fact that it controls extremely important cellular processes and, due to this, is involved in a large number of various regulatory chains.
Functional role.
The p53 protein (or its gene) is activated in response to various damage to the cellular structure:
Unrepaired breaks and other DNA damage
Violation of chromosome segregation in mitosis,
Destruction of microtubules, etc.
As a result, through the mediation of the p53 protein, the cell responds to damage to its structure
Either it lingers at one or another stage of the mitotic cycle and corrects these damages;
Or (if corrections are impossible) it stops dividing altogether and enters into the process of cellular aging;
Or (if the damaged cell is potentially dangerous to its environment) it carries out apoptosis, i.e., simply put, suicide.
In particular, cells in which tumor transformation has occurred, among others, undergo apoptosis. In this regard, it is clear why angiogenesis is simultaneously inhibited: this is another way to limit tumor growth.
Therefore, p53 protein is one of the most important tumor suppressors. In the majority of developing tumors, the functions of the p53 protein are impaired in one way or another.
5. RNA STRUCTURE
All transcription factors, like transcription itself, are designed to ensure only one thing - the formation of RNA at the required speed on certain parts of the chromosomes.
General plan of the structure of RNA
Like DNA, RNA are linear (i.e. unbranched) polynucleotides with the same principle of organization:
Consist of four types of nucleotides, each of which includes a nitrogenous base, a pentose and a phosphate residue;
Nucleotides are linked into a chain using 5´,3´-phosphodiester bonds;
Polynucleotide chains are polar, that is, they have distinguishable 5" and 3" ends.
But there are also differences from DNA. The main one is that RNA molecules (except for the RNA of some viruses) are not double-stranded, but single-stranded. The reason is the following three features of the primary structure.
a) Firstly, pentose in RNA is not deoxyribose, but ribose, which contains an additional hydroxy group. The latter makes the double-stranded structure less compact.
b) Secondly, among the four main, or major, nitrogenous bases, instead of thymine, there is uracil, which differs from thymine only in the absence of a methyl group in the 5th position.
6. RNA SYNTHESIS (DNA TRANSCRIPTION)
General characteristics of transcription
Unlike DNA replication, DNA transcription occurs in almost all nucleated cells - both dividing and non-dividing.
Moreover, in dividing cells it occurs at any moment of the mitotic cycle, except for the period of replication (in eukaryotes) and division itself.
Moreover, transcription of any section of DNA can occur not only at almost any moment in the cycle, but also repeatedly - an arbitrary number of times. On the other hand, the set of regions transcribed in a cell often changes under the influence of certain factors.
The enzymatic support of the process is carried out by RNA polymerase. Eukaryotes have three types of this enzyme:
RNA polymerase I - for the synthesis of pre-rRNA.
RNA polymerase II - for the synthesis of pre-mRNA and
RNA polymerase III - for pre-tRNA synthesis
The enzyme crawls along DNA and catalyzes the alternate incorporation into the growing chain of ribonucleotides that are complementary to the nucleotides of the DNA template chain.
Another similarity with DNA synthesis is the direction of growth of the chain being built - 5´→3´. This means that the next nucleotides of this chain are added to the 3" end.
As with all template syntheses, the strand being built is antiparallel to the DNA template strand. Consequently, the latter is transcribed by the enzyme in the 3´→5´ direction.
But there are also fundamental differences from DNA synthesis.
a) Asymmetry of the process: as we know, only one DNA strand is used as a template. It is not entirely clear how the enzyme system selects the correct chain. Apparently, the key role here is played by some nucleotide sequences on one of the chains, recognized by the system.
b) Conservative process: the DNA molecule returns to its original state upon completion of RNA synthesis. During DNA synthesis, the molecules are half renewed, which makes replication semi-conservative.
c) Finally, RNA synthesis does not require any primer to begin, whereas DNA replication requires an RNA primer.
Transcription mechanism
Initiation of transcription
The first and perhaps most important stage of transcription is its initiation: binding of RNA polymerase to the promoter and formation of the first internucleotide bond.
We have already spoken about the binding of RNA polymerase more than once, so now we will only recall the main points (with the addition of some information).
In eukaryotes Preliminary binding of a whole set of proteins of general transcription factors to the promoter is always required to form a complex. By binding to the promoter, RNA polymerase causes local denaturation of DNA, i.e., separation of DNA strands over approximately 1.5 turns of DNA. As they say, a transcriptional “eye” is formed. Thanks to this, the nucleotides of the DNA template chain in the “eye” region become available for pairing with rNTP (ribonucleoside triphosphate).
The first to be included in the RNA chain under construction is always a purine nucleotide - ATP or GTP, and all three of its phosphate residues are retained.
Then the first 5",3" phosphate bond is formed with the second nucleotide.
Transcription elongation
The next stage after initiation is elongation: the gradual lengthening of the growing pre-RNA chain to its final size.
This occurs as RNA polymerase moves along the DNA. Accordingly, the transcriptional “eye”, i.e., the area of local DNA unwinding, also moves. On the transcribed part of DNA, the double-stranded helical structure is restored immediately after the departure of RNA polymerase.
The approximate speed of enzyme movement and RNA synthesis is 30 nucleotides per second.
Termination of transcription
The last stage is termination, or the end of transcription.
The signal for this is special GC-rich regions at the end of genes. Since the interaction force between GC pairs is quite strong, local denaturation of such regions in DNA is more difficult. This slows down the progress of RNA polymerase and can serve as a signal for it to stop transcription.
But even before the end of the process, a GC-rich region also manages to appear at the end of the newly synthesized RNA. Due to the interaction between its nucleotides, it forms a “hairpin”.
That is, interactions with nucleotides of the DNA template strand are replaced by “intra-hairpin” interactions. This makes it easier for the RNA to detach from the DNA.
7. MATURATION (PROCESSING) OF RNA
Almost all RNA maturation processes can be divided into three types:
Removing some
Joining others and
Modification of the same or third nucleotides.
Removing “extra” sequences
general description
Removal of “extra” nucleotides is carried out by special nucleases. Exonucleases sequentially cleave off one nucleotide at a time from a specific end of the chain (3´ or 5´). And endonucleases cut the chain somewhere in the middle sections, leading to its fragmentation.
Mechanism of splicing
One of the key points of the mechanism under consideration is ensuring the accuracy of cutting the pre-RNA chain: an error of even one nucleotide will lead to a “frame shift”, which will change the meaning of all mRNA codons or the tRNA anticodon.
Accuracy is achieved due to two circumstances:
Firstly, at the beginning and at the end of each intron there are certain nucleotide sequences: for example, introns always begin with G-U and end with the doublet A-G.
Secondly, to recognize these sequences, special RNAs are used. small nuclear RNAs (snRNAs). The latter are associated with enzymes that catalyze splicing. Such ribonucleoprotein complexes are called spliosomes.
Splicing begins with the interaction of two snRNAs at the beginning and end of an intron. This gives an “orientation” for the endonuclease: the latter acts at the boundaries of double- and single-stranded regions.
The first pre-RNA break occurs in the region of the 5´ end of the intron - this is the location of the left edge of the left snRNA. In this case, the 5" end of the intron binds to one of the nucleotides in the middle part of the same intron, which leads to the formation of a ring structure.
Addition and modification of nucleotides
So, during the process of pre-RNA maturation, the latter loses a significant part of its nucleotides. But non-transcriptional addition of individual nucleotides also occurs.
In the case of pre-mRNA, a 7-methylguanyl nucleotide, a component of the “cap,” is attached at the 5" end (using a pyrophosphate bond, which is atypical for polynucleotides). And at the 3" end, a poly(A) fragment of approximately 200 nucleotides is expanded nucleotide-by-nucleotide. . For this purpose, special enzymes are used; in particular, for the formation of the poly(A) fragment of polyadenylate polymerase.
In the case of pre-tRNA, three nucleotides are added in turn from the 3" end - C, C and A, forming an acceptor branch.
The maturation of mRNA is called processing. The biological significance of processing in a eukaryotic cell lies in the possibility of obtaining different combinations of gene exons, and therefore obtaining a greater variety of proteins encoded by a single DNA nucleotide sequence.
In addition, modification of the 3' and 5' ends of mRNA serves to regulate its export from the nucleus, maintain stability in the cytoplasm, and improve interaction with ribosomes.
Even before transcription is completed, polyadenylation of the 3’-end occurs (Section 6.3). A 7-methylguanosine is added to the 5" end of the mRNA via a triphosphate bridge, joining at the unusual position 5"^5", and the riboses of the first two nucleotides are methylated. This process is called capping.
The process of cutting specific nucleotide sequences from RNA molecules and joining sequences retained in the “mature” molecule during RNA processing is called splicing. During splicing, sections of mRNA that do not code for proteins (introns) are removed, and exons, sections that code for an amino acid sequence, are joined to each other, and the immature pre-mRNA is converted into mature mRNA, from which cell proteins are synthesized (translated).
Splicing requires the presence of special 3" and 5" sequences. Splicing is catalyzed by a large complex of RNA and proteins called the spliceosome. The spliceosome includes five small nuclear ribonucleoproteins (snRNPs) - u1, u2, u4, u5 and ub. The RNA that is part of the snRNP interacts with the intron and may be involved in catalysis. It takes part in the splicing of introns containing GU in the 5" site and AG in the 3" splicing site.
Sometimes, during the process of maturation, mRNAs can undergo alternative splicing, which consists in the fact that the introns present in the pre-mRNA are excised in various alternative combinations, in which some exons are also excised. Some of the products of alternative splicing of pre-mRNAs are non-functional, such as in sex determination in the fruit fly Drosophila, but often alternative splicing of pre-mRNAs of a single gene produces multiple mRNAs and their protein products.
It is currently known that in humans, 94% of genes are subject to alternative splicing (the remaining 6% of genes do not contain introns). Alternative splicing in multicellular eukaryotes is a key mechanism for increasing protein diversity without creating redundant gene copies, and also allows for tissue-specific and stage-specific regulation of gene expression (manifestation).
