Aminoacyl-tRNA synthetase (ARSase) is a synthetase enzyme that catalyzes the formation of aminoacyl-tRNA in the esterification reaction of a certain amino acid with its corresponding tRNA molecule. Each amino acid has its own aminoacyl-tRNA synthetase. ARSases ensure that the nucleotide triplets of the genetic code (tRNA anticodon) correspond to the amino acids inserted into the protein, and thus ensure the correct reading of genetic information from mRNA during protein synthesis on ribosomes. Most APC-ases consist of 1, 2 or 4 identical polypeptide chains. The molecular weight of polypeptide chains is 30-140 thousand. Many APC-ases contain two active centers. There are 3 plots. The 1st site does not have specificity, it is the same for all enzymes, this is the site of ATP attachment. The nth site has strict specificity, a certain AK is attached here, according to which ARSase is called, for example, if it attaches methionine, then it is called methionyl-t-RNA synthetase. The sh-th site is also a strictly specific site, it can only connect with a certain t-RNA. Thus, the enzyme is required for the recognition of amino acids and tRNA.
The specificity of reactions catalyzed by APCases is very high, which determines the accuracy of protein synthesis in a living cell. If A. carries out an erroneous aminoacylation of tRNA with an amino acid similar in structure, a correction will occur by catalyzed by the same APC-ase hydrolysis of the erroneous AK-tRNA to AA and tRNA. The cytoplasm contains a complete set of APCases, while chloroplasts and mitochondria have their own APCases.
transport RNA. Structure, functions. The structure of the ribosome.
All tRNAs have common features both in their primary structure and in the way the polynucleotide chain is folded into a secondary structure due to interactions between the bases of nucleotide residues.
Primary structure of tRNA
tRNAs are relatively small molecules, their chain length varies from 74 to 95 nucleotide residues. All tRNAs have the same 3'-end, built from two cytosine residues and one adenosine (CCA-terminus). It is the 3'-terminal adenosine that binds to the amino acid residue during the formation of aminoacyl-tRNA. The CCA end is attached to many tRNAs by a special enzyme. The nucleotide triplet complementary to the amino acid codon (anticodon) is located approximately in the middle of the tRNA chain. The same (conservative) nucleotide residues are found in certain positions of the sequence in almost all types of tRNA. Some positions may contain either only purine or only pyrimidine bases (these are called semi-conservative residues).
All tRNA molecules are characterized by the presence of a large number (up to 25% of all residues) of various modified nucleosides, often called minor ones. They are formed at various sites in the molecules, in many cases well-defined, as a result of the modification of ordinary nucleoside residues with the help of special enzymes.
Secondary structure of tRNA
the folding of the chain into a secondary structure occurs due to the mutual complementarity of the sections of the chain. Three fragments of the chain are complementary when they are folded on themselves, forming hairpin structures. In addition, the 5" end is complementary to the site close to the 3" end of the chain, with their antiparallel arrangement; they form the so-called acceptor stem. The result is a structure characterized by the presence of four stems and three loops, which is called the "cloverleaf". A stem with a loop forms a branch. At the bottom is an anticodon branch containing an anticodon triplet as part of its loop. To its left and right are the D and T branches, respectively named for the presence of the unusual conserved dihydrouridine (D) and thymidine (T) nucleosides in their loops. The nucleotide sequences of all studied tRNAs can be folded into similar structures. In addition to the three cloverleaf loops, an additional, or variable, loop (V-loop) is also isolated in the tRNA structure. Its size differs sharply in different tRNAs, varying from 4 to 21 nucleotides, and according to recent data, up to 24 nucleotides.
Spatial (tertiary) structure of tRNA
Due to the interaction of the elements of the secondary structure, a tertiary structure is formed, which is called the L-form because of the similarity with the Latin letter L (Fig. 2 and 3). Through base stacking, the acceptor stem and cloverleaf T stem form one continuous double helix, and the other two stems form the anticodon and D stems another continuous double helix. In this case, the D- and T-loops turn out to be close and are fastened together by forming additional, often unusual base pairs. As a rule, conservative or semi-conservative residues take part in the formation of these pairs. Similar tertiary interactions also hold together some other parts of the L-structure
The main purpose of transfer RNA (tRNA) is to deliver activated amino acid residues to the ribosome and ensure their inclusion in the synthesized protein chain in accordance with the program written by the genetic code in the matrix, or information, RNA (mRNA).
The structure of the ribosome.
Ribosomes are ribonucleoprotein formations - a kind of "factory" in which amino acids are assembled into proteins. Eukaryotic ribosomes have a sedimentation constant of 80S and consist of 40S (small) and 60S (large) subunits. Each subunit includes rRNA and proteins.
Proteins are part of the subunits of the ribosome in the amount of one copy and perform a structural function, providing interaction between mRNA and tRNA associated with an amino acid or peptide.
In the presence of mRNA, the 40S and 60S subunits combine to form a complete ribosome, the mass of which is about 650 times that of the hemoglobin molecule.
Apparently, rRNA determines the main structural and functional properties of ribosomes, in particular, ensures the integrity of ribosomal subunits, determines their shape and a number of structural features.
The union of the large and small subunits occurs in the presence of messenger (messenger) RNA (mRNA). One mRNA molecule usually combines several ribosomes like a string of beads. Such a structure is called a polysome. Polysomes are freely located in the ground substance of the cytoplasm or attached to the membranes of the rough cytoplasmic reticulum. In both cases, they serve as a site for active protein synthesis.
Like the endoplasmic reticulum, ribosomes have only been discovered using an electron microscope. Ribosomes are the smallest of the cell organelles.
The ribosome has 2 centers for attaching tRNA molecules: aminoacyl (A) and peptidyl (P) centers, in the formation of which both subunits are involved. Together, the A and P centers comprise a 2-codon mRNA region. During translation, center A binds aa-tRNA, the structure of which is determined by a codon located in the region of this center. The structure of this codon encodes the nature of the amino acid that will be included in the growing polypeptide chain. The P center is occupied by peptidyl-tRNA; tRNA linked to a peptide chain that has already been synthesized.
In eukaryotes, there are 2 types of ribosomes: "free", found in the cytoplasm of cells, and associated with the endoplasmic reticulum (ER). Ribosomes associated with the ER are responsible for the synthesis of "for export" proteins that enter the blood plasma and are involved in the renewal of ER proteins, the Golgi apparatus membrane, mitochondria, or lysosomes.
Synthesis of a polypeptide molecule. initiation and elongation.
Protein synthesis is a cyclic, multi-step, energy-dependent process in which free amino acids are polymerized into a genetically determined sequence to form polypeptides.
The second stage of matrix protein synthesis, the actual translation that occurs in the ribosome, is conventionally divided into three stages: initiation, elongation, and termination.
Initiation.
A DNA sequence that is transcribed into a single mRNA, starting with a scan at the 5' end and ending with a terminator at the 3' end, is a transcription unit and corresponds to the concept of a "gene". Control of gene expression can be carried out at the stage of translation - initiation. At this stage, RNA polymerase recognizes the promoter, a 41–44 bp fragment. Transcription occurs in the 5`-3` direction or from left to right. The sequences lying to the right of the starting nucleotide, from which tRNA synthesis begins, are designated by numbers with the sign + (+1,+2..) and those to the left with the sign - (-1,-2). Thus, the region of DNA to which DNA polymerase attaches occupies an area with coordinates approximately from -20 to +20. All promoters contain the same nucleotide sequences, which are called conservative. Such sequences serve as signals recognized by RNA polymerases. The starting point is usually represented by purine. Immediately to the left of it is 6-9 bp, known as the Pribnov sequence (or box): TATAAT. It may vary somewhat, but the first two bases occur in most promoters. It is assumed that, since it is formed by a site rich in AT pairs, linked by two hydrogen bonds, DNA in this place is more easily divided into separate strands. This creates conditions for the functioning of RNA polymerase. Along with this, the Pribnov box is necessary for orientation in such a way that mRNA synthesis goes from left to right, i.e. from 5`-3`. The center of the Pribnow box is at nucleotide -10. A sequence of similar composition is located in another region centered at position 35. This 9 bp region is designated as sequence 35 or the recognition region. It is the site to which the factor attaches, thereby determining the efficiency with which RNA polymerase cannot begin transcription without special proteins. One of them is the CAP or CRP factor.
In eukaryotes, promoters interacting with RNA polymerase II have been studied in more detail. They contain three homologous regions in regions with coordinates at points -25, -27 and also at the starting point. The starting bases are adenines flanked on both sides by pyrimidines. At a distance of 19-25 b.p. to the left of the site are 7 b.p. TATAA, known as the TATA sequence, or Hogness box, is often surrounded by areas rich in GC pairs. Further to the left, at position -70 to -80, is the GTZ or CAATCT sequence, called the CAAT box. It is assumed that the TATA sequence controls the choice of the starting nucleotide, while CAAT controls the primary binding of RNA polymerase to the DNA template.
Elongation. The mRNA elongation step is similar to DNA elongation. It requires ribonucleotide triphosphates as precursors. The stage of transcription elongation, that is, the growth of the mRNA chain, occurs by attaching ribonucleotide monophosphates to the 3'-end of the chain with the release of pyrophosphate. Copying in eukaryotes usually occurs on a limited DNA region (gene), although in prokaryotes, in some cases, transcription can proceed sequentially through several linked genes that form a single operon and one common promoter. In this case, polycistronic mRNA is formed.
Regulation of gene activity on the example of the lactose operon.
The lactose operon is a bacterial polycistronic operon encoding the genes for lactose metabolism.
The regulation of lactose metabolism gene expression in Escherichia coli was first described in 1961 by scientists F. Jacob and J. Monod. The bacterial cell synthesizes the enzymes involved in the metabolism of lactose only when lactose is present in the environment and the cell lacks glucose.
The lactose operon consists of three structural genes, a promoter, an operator, and a terminator. It is assumed that the operon also includes a regulator gene that encodes a repressor protein.
Structural genes of the lactose operon - lacZ, lacY and lacA:
lacZ encodes the enzyme β-galactosidase, which breaks down the disaccharide lactose into glucose and galactose,
lacY codes for β-galactoside permease, a membrane transport protein that transports lactose into the cell.
lacA codes for β-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to beta-galactosides.
At the beginning of each operon is a special gene - the operator gene. On the structural genes of one operon, one mRNA is usually formed, and these genes are either active or inactive at the same time. As a rule, structural genes in the operon are in a state of repression.
A promoter is a DNA region recognized by the RNA polymerase enzyme, which ensures the synthesis of mRNA in the operon; it is preceded by a DNA region to which the Sar protein, an activator protein, is attached. These two sections of DNA are 85 base pairs long. After the promoter, the operon hosts the operator gene, consisting of 21 nucleotide pairs. It is usually associated with the repressor protein produced by the regulator gene. Behind the operator gene is a spacer (space-gap). Spacers are non-informative sections of a DNA molecule of various lengths (sometimes up to 20,000 base pairs), which, apparently, are involved in regulating the transcription process of an adjacent gene.
The operon ends with a terminator - a small section of DNA that serves as a stop signal for mRNA synthesis on this operon.
Acceptor genes serve as sites for attachment of various proteins that regulate the functioning of structural genes. If lactose, penetrating into the cell (in this case, it is called an inductor), blocks the proteins encoded by the regulator gene, then they lose their ability to attach to the operator gene. The gene operator goes into an active state and turns on the structural genes.
RNA polymerase, using the Cap protein (activator protein), attaches to the promoter and, moving along the operon, synthesizes pro-mRNA. During transcription, mRNA reads genetic information from all structural genes in one operon. During translation on the ribosome, the synthesis of several different polypeptide chains occurs, in accordance with the codons contained in the mRNA - nucleotide sequences that ensure the initiation and termination of the translation of each chain. The type of regulation of the work of genes, considered on the example of the lactose operon, is called the negative induction of protein synthesis.
Regulation of gene activity on the example of the tryptophan operon.
Another type of gene regulation is negative repression, studied in E.coU using the example of an operon that controls the synthesis of the tryptophone amino acid. This operon consists of 6700 base pairs and contains 5 structural genes, an operator gene and two promoters. The regulator gene ensures constant synthesis of the regulatory protein, which does not affect the functioning of the trp operon. With an excess of tryptophan in the cell, the latter combines with the regulatory protein and changes it in such a way that it binds to the operon and represses the synthesis of the corresponding mRNA.
Negative and positive control of genetic activity.
The so-called positive induction is also known, when the protein product of the regulator gene activates the work of the operon, i.e. is not a repressor, but an activator. This division is conditional, and the structure of the acceptor part of the operon, the action of the regulator gene in prokaryotes are very diverse.
The number of structural genes in the operon in prokaryotes ranges from one to twelve; An operon can have either one or two promoters and a terminator. All structural genes localized in one operon, as a rule, control a system of enzymes that provide one chain of biochemical reactions. Undoubtedly, there are systems in the cell that coordinate the regulation of the work of several operons.
Proteins that activate mRNA synthesis are attached to the first part of the gene acceptor - operator, and proteins - repressors that suppress mRNA synthesis are attached to its end. One gene is regulated by one of several proteins, each of which attaches to a corresponding acceptor site. Different genes can have common regulators and identical operator regions. Regulatory genes do not act simultaneously. First, one immediately includes one group of genes, then after a while the other - another group, i.e. the regulation of gene activity occurs in "cascades", and the protein synthesized in one stage can be a regulator of protein synthesis in the next stage.
The structure of chromosomes. Karyotype. Idiogram. Models of the structure of chromosomes.
Eukaryotic chromosomes are complex. The basis of the chromosome is a linear (not closed in a ring) macromolecule of deoxyribonucleic acid (DNA) of considerable length (for example, in the DNA molecules of human chromosomes, there are from 50 to 245 million pairs of nitrogenous bases). In a stretched form, the length of a human chromosome can reach 5 cm. In addition to it, the chromosome includes five specialized proteins - H1, H2A, H2B, H3 and H4 (the so-called histones) and a number of non-histone proteins. The amino acid sequence of histones is highly conserved and practically does not differ in various groups of organisms. In interphase, chromatin is not condensed, but even at this time its threads are a complex of DNA and proteins. Chromatin is a deoxyribonucleoprotein that can be seen under a light microscope in the form of thin filaments and granules. A DNA macromolecule wraps around octomers (structures consisting of eight protein globules) of histone proteins H2A, H2B, H3 and H4, forming structures called nucleosomes.
In general, the whole design is somewhat reminiscent of beads. A sequence of such nucleosomes connected by an H1 protein is called a nucleofilament, or nucleosomal filament, with a diameter of about 10 nm.
The condensed chromosome looks like an X (often with unequal arms) because the two chromatids resulting from replication are still connected to each other at the centromere. Each cell of the human body contains exactly 46 chromosomes. Chromosomes are always in pairs. A cell always has 2 chromosomes of each species, pairs differ from each other in length, shape and the presence of thickenings or constrictions.
Centromere - a specially organized section of the chromosome, common to both sister chromatids. The centromere divides the body of the chromosome into two arms. Depending on the location of the primary constriction, the following types of chromosomes are distinguished: equal-arm (metacentric), when the centromere is located in the middle, and the arms are approximately equal in length; unequal arms (submetacentric), when the centromere is displaced from the middle of the chromosome, and the arms are of unequal length; rod-shaped (acrocentric), when the centromere is shifted to one end of the chromosome and one arm is very short. In some chromosomes, there may be secondary constrictions that separate a region called the satellite from the body of the chromosome.
The study of the chemical organization of the chromosomes of eukaryotic cells showed that they consist mainly of DNA and proteins. As has been proven by numerous studies, DNA is a material carrier of the properties of heredity and variability and contains biological information - a program for the development of a cell, an organism, written using a special code. Proteins make up a significant part of the substance of chromosomes (about 65% of the mass of these structures). The chromosome, as a complex of genes, is an evolutionarily established structure that is characteristic of all individuals of a given species. The mutual arrangement of genes in the chromosome plays an important role in the nature of their functioning.
A graphic representation of a karyotype showing its structural features is called an idiogram.
A set of chromosomes specific to a certain species in number and structure is called a karyotype.
Histones. Structure of nucleosomes.
Histones are the main class of nucleoproteins, the nuclear proteins required for the assembly and packaging of DNA strands into chromosomes. There are five different types of histones, named H1/H5, H2A, H2B, H3, H4. The sequence of amino acids in these proteins practically does not differ in organisms of different levels of organization. Histones are small, strongly basic proteins that bind directly to DNA. Histones take part in the structural organization of chromatin, neutralizing the negatively charged phosphate groups of DNA due to the positive charges of amino acid residues, which makes possible the dense packing of DNA in the nucleus.
Two molecules of each of the histones H2A, H2B, H3, and H4 make up an octamer entwined with a 146 bp DNA segment, forming 1.8 turns of the helix over the protein structure. This particle with a diameter of 7 nm is called the nucleosome. A section of DNA (linker DNA) that is not directly in contact with the histone octamer interacts with histone H1.
The group of non-histone proteins is highly heterogeneous and includes structural nuclear proteins, many enzymes and transcription factors associated with certain DNA regions and regulating gene expression and other processes.
The histones in the octamer have a mobile N-terminal fragment ("tail") of 20 amino acids, which protrudes from the nucleosomes and is important for maintaining chromatin structure and controlling gene expression. So, for example, the formation (condensation) of chromosomes is associated with phosphorylation of histones, and the enhancement of transcription is associated with acetylation of lysine residues in them. The details of the mechanism of regulation have not been fully elucidated.
Nucleosome is a chromatin subunit consisting of DNA and a set of four pairs of histone proteins H2A, H2B, H3 and H4 of one H1 histone molecule. Histone H1 binds to the linker DNA between two nucleosomes.
The nucleosome is the basic unit of chromatin packaging. It consists of a DNA double helix wrapped around a specific complex of eight nucleosome histones (the histone octamer). The nucleosome is a disc-shaped particle with a diameter of about 11 nm, containing two copies of each of the nucleosomal histones (H2A, H2B, H3, H4). The histone octamer forms a protein core around which is double-stranded DNA (146 nucleotide pairs of DNA per histone octamer).
The nucleosomes that make up the fibrils are located more or less evenly along the DNA molecule at a distance of 10–20 nm from each other.
Packing levels of eukaryotic chromosomes. chromatin condensation.
Thus, the DNA packaging levels are as follows:
1) Nucleosomal (2.5 turns of double-stranded DNA around eight molecules of histone proteins).
2) Supernucleosomal - chromatin helix (chromonema).
3) Chromatid - spiralized chromonema.
4) Chromosome - the fourth degree of DNA spermalization.
In the interphase nucleus, the chromosomes are decondensed and are represented by chromatin. The despiralized region containing genes is called euchromatin (loose, fibrous chromatin). This is a necessary condition for transcription. During rest between divisions, certain sections of chromosomes and entire chromosomes remain compact.
These spiralized, strongly stained areas are called heterochromatin. They are inactive for transcription. There are facultative and constitutive heterochromatin.
Facultative heterochromatin is informative, because contains genes and can pass into euchromatin. Of the two homologous chromosomes, one may be heterochromatic. Constitutive heterochromatin is always heterochromatic, non-informative (does not contain genes), and therefore is always inactive in relation to transcription.
Chromosomal DNA consists of more than 108 base pairs, from which informative blocks are formed - genes arranged linearly. They account for up to 25% of DNA. A gene is a functional unit of DNA containing information for the synthesis of polypeptides, or all RNA. Between the genes are spacers - non-informative segments of DNA of different lengths. Excess genes are represented by a large number - 104 identical copies. An example are genes for t-RNA, r-RNA, histones. In DNA, there are sequences of the same nucleotides. They can be moderately repetitive and highly repetitive sequences. Moderately repetitive sequences reach 300 base pairs with repetitions 102 - 104 and most often represent spacers, redundant genes.
Highly repetitive sequences (105 - 106) form constitutive heterochromatin. About 75% of all chromatin is not involved in transcription, it falls on highly repetitive sequences and non-transcribed spacers.
Preparation of chromosome preparations. The use of colchicine. Hypotonia, fixation and staining.
Depending on the degree of proliferative activity of cells of different tissues in vivo and in vitro, direct and indirect methods for obtaining chromosome preparations are distinguished.
1) Direct methods are used in the study of tissues with high mitotic activity (bone marrow, chorion and placenta, cells of the lymph nodes, tissues of the embryo at an early stage of development). Chromosome preparations are prepared directly from freshly obtained material after special processing.
2) Indirect methods include obtaining chromosome preparations from any tissue after its preliminary cultivation for a different period of time.
There are many modifications of direct and indirect methods for preparing chromosome preparations, however, the main steps for obtaining metaphase plates remain unchanged:
1. The use of colchicine (colcemid) - an inhibitor of the formation of the mitotic spindle, which stops cell division at the metaphase stage.
2. Hypotonic shock with the use of solutions of potassium or sodium salts, which, due to the difference in osmotic pressure inside and outside the cells, cause them to swell and break interchromosomal bonds. This procedure leads to the separation of chromosomes from each other, contributing to their greater spread in the metaphase plates.
3. Fixation of cells using glacial acetic acid and ethanol (methanol) in a ratio of 3:1 (Carnoy's fixative), which contributes to the preservation of the chromosome structure.
4. Dropping the cell suspension onto glass slides.
5. Staining of chromosome preparations.
A number of staining (banding) methods have been developed that make it possible to identify a complex of transverse marks (bands, bands) on a chromosome. Each chromosome is characterized by a specific set of bands. Homologous chromosomes stain identically, with the exception of polymorphic regions where different allelic variants of genes are localized. Allelic polymorphism is characteristic of many genes and is found in most populations. Detection of polymorphisms at the cytogenetic level has no diagnostic value.
A. Q-staining. The first method of differential staining of chromosomes was developed by the Swedish cytologist Kaspersson, who used for this purpose the fluorescent dye acrichin mustard. Under a fluorescent microscope on the chromosomes are visible areas with unequal fluorescence intensity - Q-segments. The method is best suited for the study of Y chromosomes and therefore is used to quickly determine the genetic sex, identify translocations (site exchanges) between the X and Y chromosomes or between the Y chromosome and autosomes, as well as to view a large number of cells when it is necessary to find out whether a patient with mosaicism on the sex chromosomes has a clone of cells carrying the Y chromosome.
B. G-staining. After extensive pretreatment, often with trypsin, the chromosomes are stained with Giemsa stain. Under a light microscope, light and dark stripes are visible on the chromosomes - G-segments. Although the arrangement of the Q segments corresponds to that of the G segments, G staining has proven to be more sensitive and has taken the place of Q staining as the standard method of cytogenetic analysis. G-staining gives the best results in detecting small aberrations and marker chromosomes (segmented differently than normal homologous chromosomes).
B. R-staining gives a picture opposite to G-staining. Usually Giemsa stain or acridine orange fluorescent stain is used. This method reveals differences in staining of homologous G- or Q-negative regions of sister chromatids or homologous chromosomes.
D. C-staining is used to analyze the centromeric regions of chromosomes (these regions contain constitutive heterochromatin) and the variable, brightly fluorescent distal portion of the Y chromosome.
E. T-staining is used to analyze telomeric regions of chromosomes. This technique, as well as staining of regions of nucleolar organizers with silver nitrate (AgNOR-staining) is used to refine the results obtained by standard staining of chromosomes.
The interaction and structure of IRNA, tRNA, RRNA - the three main nucleic acids, is considered by such a science as cytology. It will help to find out what is the role of transport (tRNA) in cells. This very small, but at the same time undeniably important molecule takes part in the process of combining the proteins that make up the body.
What is the structure of tRNA? It is very interesting to consider this substance "from the inside", to find out its biochemistry and biological role. And also, how are the structure of tRNA and its role in protein synthesis interrelated?
What is TRNA, how is it arranged?
Transport ribonucleic acid is involved in the construction of new proteins. Almost 10% of all ribonucleic acids are transport. To make it clear what chemical elements a molecule is formed from, we will describe the structure of the secondary structure of tRNA. The secondary structure considers all the major chemical bonds between the elements.
Consisting of a polynucleotide chain. Nitrogenous bases in it are connected by hydrogen bonds. Like DNA, RNA has 4 nitrogenous bases: adenine, cytosine, guanine, and uracil. In these compounds, adenine is always associated with uracil, and guanine, as usual, with cytosine.
Why does a nucleotide have the prefix ribo-? Simply, all linear polymers that have a ribose instead of a pentose at the base of the nucleotide are called ribonucleic. And transfer RNA is one of 3 types of just such a ribonucleic polymer.
The structure of tRNA: biochemistry
Let's look into the deepest layers of the structure of the molecule. These nucleotides have 3 components:
- Sucrose, ribose is involved in all types of RNA.
- Phosphoric acid.
- nitrogenous and pyrimidines.
Nitrogenous bases are linked together by strong bonds. It is customary to divide bases into purine and pyrimidine.
Purines are adenine and guanine. Adenine corresponds to an adenyl nucleotide of 2 interconnected rings. And guanine corresponds to the same “single-ring” guanine nucleotide.
Pyramidines are cytosine and uracil. Pyrimidines have a single ring structure. There is no thymine in RNA, since it is replaced by an element such as uracil. This is important to understand before looking at other structural features of tRNA.
Types of RNA
As you can see, the structure of tRNA cannot be briefly described. You need to delve into biochemistry to understand the purpose of the molecule and its true structure. What other ribosomal nucleotides are known? There are also matrix or informational and ribosomal nucleic acids. Abbreviated as RNA and RNA. All 3 molecules work closely with each other in the cell so that the body receives correctly structured protein globules.
It is impossible to imagine the work of one polymer without the help of 2 others. Structural features of tRNAs become more understandable when considered in conjunction with functions that are directly related to the work of ribosomes.
The structure of RNA, tRNA, rRNA is similar in many ways. All have a ribose base. However, their structure and functions are different.
Discovery of nucleic acids
The Swiss Johann Miescher found macromolecules in the cell nucleus in 1868, later called nucleins. The name "nucleins" comes from the word (nucleus) - the nucleus. Although a little later it was found that in unicellular creatures that do not have a nucleus, these substances are also present. In the middle of the 20th century, the Nobel Prize was received for the discovery of the synthesis of nucleic acids.
in protein synthesis
The name itself - transfer RNA - indicates the main function of the molecule. This nucleic acid "brings" with it the essential amino acid required by the ribosomal RNA to make a particular protein.
The tRNA molecule has few functions. The first is the recognition of the IRNA codon, the second function is the delivery of building blocks - amino acids for protein synthesis. Some more experts distinguish the acceptor function. That is, the addition of amino acids according to the covalent principle. It helps to “attach” this amino acid to an enzyme such as aminocil-tRNA synthatase.
How is the structure of tRNA related to its functions? This special ribonucleic acid is designed in such a way that on one side of it there are nitrogenous bases, which are always connected in pairs. These are the elements known to us - A, U, C, G. Exactly 3 "letters" or nitrogenous bases make up the anticodon - a reverse set of elements that interacts with the codon according to the principle of complementarity.
This important structural feature of tRNA ensures that there will be no errors in decoding the template nucleic acid. After all, it depends on the exact sequence of amino acids whether the protein that the body needs at the present time is synthesized correctly.
Structural features
What are the structural features of tRNA and its biological role? This is a very ancient structure. Its size is somewhere around 73 - 93 nucleotides. The molecular weight of the substance is 25,000-30,000.
The structure of the secondary structure of tRNA can be disassembled by examining the 5 main elements of the molecule. So, this nucleic acid consists of the following elements:
- loop for contact with the enzyme;
- loop for contact with the ribosome;
- anticodon loop;
- acceptor stem;
- the anticodon itself.
And also allocate a small variable loop in the secondary structure. One arm in all types of tRNA is the same - a stem of two cytosine and one adenosine residues. It is in this place that the connection with 1 of the 20 available amino acids occurs. For each amino acid, a separate enzyme is intended - its own aminoacyl-tRNA.
All the information that encrypts the structure of all is contained in the DNA itself. The structure of tRNA in all living creatures on the planet is almost identical. It will look like a leaf when viewed in 2-D.
However, if you look in volume, the molecule resembles an L-shaped geometric structure. This is considered the tertiary structure of tRNA. But for the convenience of studying it is customary to visually “untwist”. The tertiary structure is formed as a result of the interaction of the elements of the secondary structure, those parts that are mutually complementary.
The tRNA arms or rings play an important role. One arm, for example, is required for chemical bonding with a particular enzyme.
A characteristic feature of a nucleotide is the presence of a huge number of nucleosides. There are more than 60 types of these minor nucleosides.
tRNA structure and amino acid coding
We know that the tRNA anticodon is 3 molecules long. Each anticodon corresponds to a specific, "personal" amino acid. This amino acid is connected to the tRNA molecule using a special enzyme. As soon as the 2 amino acids come together, the bonds to the tRNA are broken. All chemical compounds and enzymes are needed until the required time. This is how the structure and functions of tRNA are interconnected.
In total, there are 61 types of such molecules in the cell. There can be 64 mathematical variations. However, 3 types of tRNA are absent due to the fact that exactly this number of stop codons in IRNA does not have anticodons.
Interaction between RNA and tRNA
Let us consider the interaction of a substance with RNA and RRNA, as well as structural features of tRNA. The structure and purpose of a macromolecule are interrelated.
The structure of the IRNA copies information from a separate section of DNA. DNA itself is too large a connection of molecules, and it never leaves the nucleus. Therefore, an intermediary RNA is needed - informational.
Based on the sequence of molecules copied by the RNA, the ribosome builds a protein. The ribosome is a separate polynucleotide structure, the structure of which needs to be explained.
Ribosomal tRNA: interaction
Ribosomal RNA is a huge organelle. Its molecular weight is 1,000,000 - 1,500,000. Almost 80% of the total amount of RNA is precisely ribosomal nucleotides.
It seems to capture the IRNA chain and wait for anticodons that will bring tRNA molecules with them. Ribosomal RNA consists of 2 subunits: small and large.
The ribosome is called the "factory", because in this organelle all the synthesis of the substances necessary for everyday life takes place. It is also a very ancient cell structure.
How does protein synthesis occur in the ribosome?
The structure of tRNA and its role in protein synthesis are interrelated. The anticodon located on one of the sides of the ribonucleic acid is suitable in its form for the main function - the delivery of amino acids to the ribosome, where the phased alignment of the protein takes place. Essentially, the TRNA acts as an intermediary. Its task is only to bring the necessary amino acid.
When information is read from one part of the RNA, the ribosome moves further along the chain. The template is needed only to convey encoded information about the configuration and function of a single protein. Next, another tRNA approaches the ribosome with its nitrogenous bases. It also decodes the next part of the MRNA.
Decoding proceeds as follows. Nitrogenous bases combine according to the principle of complementarity in the same way as in DNA itself. Accordingly, TRNA sees where it needs to "moor" and to which "hangar" to send the amino acid.
Then, in the ribosome, the amino acids selected in this way are chemically bound, step by step a new linear macromolecule is formed, which, after the end of synthesis, twists into a globule (ball). Used tRNA and RNA, having fulfilled their function, are removed from the protein "factory".
When the first part of the codon joins with the anticodon, the reading frame is determined. Subsequently, if for some reason a frame shift occurs, then some sign of the protein will be rejected. The ribosome cannot intervene in this process and solve the problem. Only after the process is completed, the 2 rRNA subunits are combined again. On average, for every 10 4 amino acids, there is 1 error. For every 25 proteins already assembled, at least 1 replication error is sure to occur.
tRNA as relic molecules
Since tRNA may have existed at the time of the birth of life on earth, it is called a relic molecule. It is believed that RNA is the first structure that existed before DNA and then evolved. The RNA World Hypothesis - formulated in 1986 by laureate Walter Gilbert. However, it is still difficult to prove this. The theory is defended by obvious facts - tRNA molecules are able to store blocks of information and somehow implement this information, that is, perform work.
But opponents of the theory argue that a short period of the life of a substance cannot guarantee that tRNA is a good carrier of any biological information. These nucleotides are rapidly degraded. The lifetime of tRNA in human cells ranges from several minutes to several hours. Some species can last up to a day. And if we talk about the same nucleotides in bacteria, then the terms are much shorter - up to several hours. In addition, the structure and functions of tRNA are too complex for a molecule to become the primary element of the Earth's biosphere.
This article is the second in a series of auto-publishing, which must be read after reading the first article.Properties of the genetic code - a trace of its occurrence . It is highly desirable for people who are new to the basics of molecular biology to read the article by O.O. Favorova " ". It is important to understand, in order to understand HOW genetic code, it is necessary to understand HOW it functions in modern organisms. And for this it is necessary to delve into the molecular mechanisms of encoded protein synthesis. To understand this article, it is important to understand how the RNA molecule is arranged, how it differs from the DNA molecule.
Understanding the topic of the origin of life in general, and the emergence of the genetic code, in particular, is simply impossible without understanding the basic molecular mechanisms in living organisms, primarily two aspects - the reproduction of hereditary molecules (nucleic acids) and protein synthesis. Therefore, this article is devoted primarily to the presentation of that minimum of knowledge with which one can understand the rich and rather interesting material related to the origin of the genetic code (GC).
It is best to start your acquaintance with the molecular mechanisms of protein synthesis by studying the structure of one of the key components and one of the most ancient structures in living organisms - the transfer RNA (or tRNA) molecule. The tRNA molecule has an unusually conserved structure, which is similar in all living organisms. This structure changes in the course of evolution so slowly that it allows us to extract a lot of information about how the oldest protein-synthesizing systems could look like during their initial formation. Therefore, the tRNA molecule is said to bemolecular relic.
Molecular relic, or molecular fossil is an abstraction denoting ancient mechanisms and molecular and supramolecular structures found in modern organisms, which allows us to extract information about the structure of the oldest living systems. Molecular relics include molecules of ribosomal and transfer RNA, aminoacyl-tRNA synthetases, DNA and RNA polymerases, and genetic code, as a way of coding, as well as a number of other molecular structures and mechanisms. Their analysis is a key source of information about how life could have arisen, and genetic code, in particular. Let us consider in more detail the structure of tRNA and those parts of it that change so slowly during evolution that they still contain a lot of information about ancient tRNAs that existed more than 3.5 billion years ago.
The tRNA molecule is relatively small, its length varies from 74 to 95 nucleotide residues, most often 76 nucleotides (see Fig. 1).In the tRNA sequence, the so-calledconservative nucleotide residues are nucleotide residues located in strictly defined sequences in almost all tRNA molecules. In addition, stand outsemi-conservative nucleotide residues are residues represented only by purine or pyrimidine bases in strictly defined tRNA sequences. In addition, different regions of tRNA change at significantly different rates.
Up to 25% of all nucleotide residues are modified nucleosides, often referred to as minor . More than 60 minor residues have already been described. They are formed as a result of the modification of ordinary nucleoside residues with the help of special enzymes.
Pseudouridine (5-ribofuranosyluracil, Ψ), 5,6-dihydrouridine (D), 4-thiouridyl and inosine. The structure of some modified bases and partly their role are described in the article
Along with the primary structure (it's just a sequence of nucleotides), the tRNA molecule has a secondary and tertiary structure.
The secondary structure is due to the formation of hydrogen bonds between nucleotides. Even at school, they teach about hydrogen bonds during complementary pairing between nucleotides (AU and GC this type of pairing of nucleotides is called canonical), but a considerable number of non-canonical bonds are also formed in tRNA molecules, in particular, between G and U, which will be somewhat weaker and energetically less advantageous).
Rice. 1. Generalized secondary structure of tRNA (left) and generally accepted nucleotide numbering in tRNA (right). This is how it looks in almost all living organisms. In the right figure, conservative nucleotides are highlighted in bold circles.
Designations:N - any nucleotide, T - thymine, D - dihydrouridine, Ψ - pseudouridine, R - purine nucleotide.
As a result, the so-called cloverleaf structure is formed.In the structure of a clover leaf, there are: an acceptor stem and three branches, or domains (arms): anticocodon (consists of an anticodon double-stranded stem (stem) and anticodon loop (loop), dihydrouridine, orD- branch, orD-domain, (also from dihydrouridine loop and stem) andTΨC-branch, or simply T-branch, or T-domain, (T-loop and T-stem). In addition to the three cloverleaf loops, there is also a so-called additional or variable loop. The length of the variable loop varies from 4 to 24 nucleotides.
Why does the secondary structure of tRNA have a cloverleaf shape? The answer to this question was given by M. Eigen [Eigen M, Winkler R.1979] . The fact is thatwith an RNA chain length of 80 nucleotides with a random sequence, a secondary structure with 3-4 petals is the most probable. Although a hairpin with only one loop has the maximum number of base pairings, this structure in random sequences is unlikely. That is why it is reasonable to consider that tRNA-like structures (that is, structures with 3-4 loops) were the most common molecules at the stage of RNA and RNA-protein life. Additional arguments in favor of this statement will be given in the following articles.
Tertiary structure of tRNA.
The tertiary structure of tRNA corresponds to the real spatial structure. She got the nameL-forms, due to the similarity of the tertiary structure to the form of the Latin capital letter "L". The tertiary structure is formed due to the interaction of the elements of the secondary structure. Participate in its formation staking interactions grounds. Due to stacking of bases, the acceptor and T-stem of the cloverleaf form one continuous double helix, forming one of the "rods"L-forms. Anticodon andD- stems form another "stick" of this letter,D- andT-loops in such a structure turn out to be close and are fastened together by forming additional, often unusual base pairs, which, as a rule, are formed by conservative or semi-conservative residues. In light of this involvement of conservative and semi-conservative foundations in educationL-forms become clear their presence inT- andD-loops. The formation of the L-shaped structure and its interaction with APCase is shown schematically in fig. 2.
Rice. 2.Spatial education schemeL-shaped structure of tRNA and its interaction with ARSase oh.
The arrow indicates the site of attachment of the amino acid during aminoacylation of tRNA synthetase. The tRNA acceptor domain is highlighted in red, the anticodon domain is highlighted in blue. The ovals indicate the APCase domains: green is the catalytic domain containing the binding and aminoacylation domain of the tRNA acceptor region, yellow and orange are the variable domain of APCase. Depending on the size of this domain, APCase a recognizes the anticodon region as a variable domain (domain is indicated in yellow), or does not recognize it (domain is indicated in orange).
The bases of the anticodon are reversedinside L-shaped molecule.
Transfer RNAs in all living organisms sequentially perform three functions necessary for protein synthesis:
1) acceptor - with the help of protein enzymes (aminoacyl-tRNA-syntatase) covalently attaches a strictly defined amino acid to the aminoacyl residue (for each amino acid - strictly its own one or sometimes several different tRNAs);2) transport - transports an amino acid to a specific location on the ribosome;3) adaptive - in combination with the ribosome, it is able to specifically recognize the triplet of the genetic code on matrix RNA, after which the amino acid attached to the tRNA is included in the growing polypeptide chain on the ribosome.
Articles related to the topic:
The structure of transfer RNAs and their function at the first (pre-ribosomal) stage of protein biosynthesis
70-90N | secondary page - cloverleaf | CCA 3" const for all tRNA |
the presence of thymine, pseudouridine-psi, digirouridine DGU in the D-loop - protection against ribonucleases? long-lived | A variety of primary structures of tRNA - 61 + 1 - by the number of codons + formylmethionine tRNA, the cat's anticodon is the same as that of methionine tRNA. Variety of tertiary structures - 20 (according to the number of amino acids) | recognition - the formation of a covalent bond m-y tRNA and act | aminoacyl-tRNA synthetases attach acts to tRNA
The function of tRNA is to transfer amino acids from the cytoplasm to the ribosomes, in which protein synthesis occurs.
tRNAs that bind one amino acid are called isoacceptor.
In total, 64 different tRNAs simultaneously exist in a cell.
Each tRNA pairs only with its own codon.
Each tRNA recognizes its own codon without the involvement of an amino acid. The amino acids bound to the tRNA were chemically modified, after which the resulting polypeptide, which contained the modified amino acid, was analyzed. Cysteinyl-tRNACys (R=CH2-SH) was reduced to alanyl-tRNACys (R=CH3).
Most tRNAs, regardless of their nucleotide sequence, have a cloverleaf-shaped secondary structure due to the presence of three hairpins in it.
Structural features of tRNA
There are always four unpaired nucleotides at the 3 "end of the molecule, and three of them are necessarily CCAs. The 5" and 3" ends of the RNA chain form an acceptor stem. The chains are held together due to the complementary pairing of seven nucleotides 5" - end with seven nucleotides located near the 3 "end. 2. All molecules have a T? C hairpin, so designated because it contains two unusual residues: ribothymidine (T) and pseudouridine (? The hairpin consists of a double-stranded stem of five base pairs, including a pair of GCs, and a loop of seven nucleotides in length.
at the same point in the loop. 3. In an anticodon hairpin, the stem is always represented by a family of paired
grounds. The triplet complementary to the related codon, the anticodon, is located in the loop.
le, consisting of seven nucleotides. An invariant ura-
cyl and a modified cytosine, and a modified purine adjoins its 3 "end, as a rule
adenine. 4. Another hairpin consists of a stalk three to four pairs of nucleotides long and a variable loop
size, often containing uracil in a reduced form - dihydrouracil (DU). The nucleotide sequences of the stems, the number of nucleotides between the anticodon stem and the T?C stem (variable loop), as well as the size of the loop and the localization of dihydrouracil residues in the DU loop vary most strongly.
[Singer, 1998].
Tertiary structure of tRNA
L-shaped structure.
Attachment of amino acids to tRNA
In order for an amino acid to form a polypeptide chain, it must be attached to tRNA by the enzyme aminoacyl-tRNA synthetase. This enzyme forms a covalent bond between the amino acid carboxyl group and the ribose hydroxyl group at the 3' end of tRNA with the participation of ATP. Aminoacyl-tRNA synthetase recognizes a specific codon not because of the presence of an anticodon on the tRNA, but by the presence of a specific recognition site on the tRNA.
In total, there are 21 different aminoacyl-tRNA synthetases in the cell.
Joining takes place in two stages:
1. The carboxyl group of an amino acid is attached to ATP a-phosphate. The resulting unstable aminoacyl adenylate is stabilized by binding to the enzyme.
2. Transfer of the aminoacyl group of aminoacyl adenylate to the 2' or 3'-OH group of the terminal ribose of tRNA
Some aminoacyl-tRNA synthetases consist of a single polypeptide chain, while others consist of two or four identical chains, each with a molecular weight of 35 to 115 kDa. Some dimeric and tetrameric enzymes are composed of two types of subunits. There is no clear correlation between the size of the enzyme molecule or the nature of its subunit structure and specificity.
The specificity of an enzyme is determined by its strong binding to the acceptor end of tRNA, the DU region, and the variable loop. Some enzymes do not seem to recognize the anticodon triplet and catalyze the aminoacetylation reaction even when the anticodon is altered. However, some enzymes show reduced activity in relation to such modified tRNAs and add the wrong amino acid when replacing the anticodon.
70-90n | secondary page - cloverleaf | CCA 3" const for all tRNA |
the presence of thymine, pseudouridine-psi, digirouridine DGU in the D-loop - protection against ribonucleases? long-lived | A variety of primary structures of tRNA - 61 + 1 - by the number of codons + formylmethionine tRNA, the cat's anticodon is the same as that of methionine tRNA. Variety of tertiary structures - 20 (according to the number of amino acids)
There are two types of tRNA binding methionine tRNAFMet and tRNAMMet in prokaryotes and tRNAIMet and tRNAMMet in eukaryotes. Methionine is added to each tRNA using the appropriate aminoacyl-tRNA synthesis. methionine attached to tRNAFMet and tRNAIMet is formed by the enzyme methionyl-tRNA-transformylase to Fmet-tRNAFMet. tRNAs loaded with formylmethionine recognize the initiation codon AUG.
Literature:
Unfortunately, there is no bibliography.
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.
tRNA molecules are polynucleotide chains synthesized on specific DNA sequences. They consist of a relatively small number of nucleotides -75-95. As a result of the complementary connection of bases that are located in different parts of the tRNA polynucleotide chain, it acquires a structure resembling a clover leaf in shape (Fig. 3.26).
Rice. 3.26. The structure of a typical tRNA molecule.
It has four main parts that perform different functions. acceptor The "stalk" is formed by two complementary connected terminal parts of the tRNA. It consists of seven base pairs. The 3" end of this stem is somewhat longer and forms a single-stranded region that terminates in a CCA sequence with a free OH group. A transportable amino acid is attached to this end. The remaining three branches are complementary-paired nucleotide sequences that terminate in unpaired loop-forming regions. The middle of of these branches - 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.
Between the acceptor and anticodon branches are two side branches. In their loops, they contain modified bases - dihydrouridine (D-loop) and the TψC triplet, where \y is pseudouriain (T^C-loop).
Between the aiticodone and T^C branches there is an additional loop, which includes from 3-5 to 13-21 nucleotides.
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 causes a three-dimensional tertiary structure, which is characterized by the formation of two perpendicular double helices (Fig. 3.27). 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.
Rice. 3.27. Spatial organization of tRNA:
I - the secondary structure of tRNA in the form of a "clover leaf", determined by its primary structure (the sequence of nucleotides in the chain);
II - two-dimensional projection of the tertiary structure of tRNA;
III - layout of the tRNA molecule in space
APPENDIX (in case someone does not understand this)
Lightning teeth - nucleotides (Adenine-Thymine / Uracil /, Guanine-Cytazine). All lightning is DNA.
To transfer information from DNA, you need to break 2 strands. The bond between A-T and G-C is hydrogen, therefore it is easily broken by the Helicase enzyme:
To prevent knots from forming (As an example, I twisted a towel):
Topoisomerase cuts one strand of DNA at the origin of replication so that the chain does not twist.
When one thread is free, the second can easily rotate around its axis, thereby relieving tension during "untwisting". Nodes do not appear, energy is saved.
Then, an RNA primer is needed to start collecting RNA. A protein that assembles mRNA cannot just assemble the first nucleotide, it needs a piece of RNA to start (it’s written in detail there, I’ll write it out later). This piece is called the RNA primer. And this protein already attaches the first nucleotide to it.
