Cellular conveyor belt during protein synthesis. Research methods in histology
With the emergence of compartments, the eukaryotic cell receives not only obvious advantages, but also a number of problems. One of them is the sorting and delivery of the right compounds to the right organelles. First of all, this concerns proteins. The fate of synthesized proteins is different and depends on the sites of their subsequent functioning. There are two main pathways for protein transport, which begin in different places in the cytoplasm. Rice. 1.2.
The first transport branch works with proteins that are intended for plastids, mitochondria, the nucleus and peroxisomes - that is, for all cell compartments except the organelles of the endomembrane system. The synthesis of these proteins occurs on free ribosomes in the cytosol. Proteins intended for transport contain sorting signals that direct them to the appropriate organelles. Such signals are usually served by one or more sections of the protein, which are called signal or leader peptides. In the membrane of the organelle there is a special translocator protein that “recognizes” the signal peptide. The transported protein molecule must unfold in order to, like the thread of an unfolded ball, “thread” through the “eye of a needle” of the translocator protein. Table 1.1. Some characteristics of various sorting signals are presented. This protein transport pathway is sometimes called cytosolic. It should be noted that most proteins synthesized on free ribosomes in the cytosol do not have sorting signals and remain in the cytosol as permanent components.
Another transport branch is used for secreted proteins, as well as for proteins destined for organelles of the endomembrane system and the plasma membrane. The synthesis of these proteins also begins on cytosolic ribosomes, but after initiation of translation, the ribosomes attach to the ER membrane, and a rough ER is formed. The resulting proteins are transferred to the ER cotranslationally. This means that immediately after the synthesis of the next section of the polypeptide chain, it crosses the ER membrane. After synthesis, some of the proteins enter the lumen of the ER, others remain anchored in the membrane and become transmembrane ER proteins. This transport branch is often called secretory route cells.
Table 1.1. Signal sequences for protein transport in plant cells.
Target organelle | Signal sequence | Characteristic |
Chloroplasts: stroma | N-terminal leader peptide ("stromal") | Sequence of 40-50 amino acids |
Chloroplasts: lumen and thylakoid membranes | Two consecutive N-terminal leader peptides | The first peptide is “stromal”, the second is “lumenal” |
Mitochondria: matrix | N-terminal presequence | Forms a positively charged amphipathic α-loop. |
Mitochondria: inner membrane, intermembrane space | Two consecutive N-terminal presequences | The first presequence is the same as for matrix proteins, the second consists of hydrophobic amino acid residues |
Peroxisomes | Peroxisomal localization signals PTS1 and PTS2 | PTS1 – C-terminal tripeptide – Ser-Lys-Leu PTS2 is localized at the N-terminus. |
Core | NLS nuclear localization signals. Not cleaved after protein transfer to the nucleus | NLS type 1: Pro-Lys-Lys-Lys-Arg-Lys. NLS type 2: two sequences separated by a spacer NLS type 3: Lys-Ile-Pro-Ile-Lys |
Secretory pathway signal peptide | N-terminal leader peptide | 10-15 hydrophobic amino acid residues forming an α-helix. |
Endoplasmic reticulum | Localization signal in the ER | C-terminal tetrapeptide KDEL (Lys-Asp-Glu-Leu) |
Vacuole. | Localization signals in vacuoles NTPP, CTPP, intraprotein signal. | NTPP - N-terminal signal: Asn-Pro-lle-Arg CTPP - C-terminal signal. |
The two branches of transport differ in their transportation patterns. The paths of cytosolic transport of proteins are parallel, that is, proteins from the cytosol are immediately sent to the desired organelle. Typically, no more than one to two minutes pass from the moment the protein is released into the cytosol until it enters the organelle.
Transport of proteins along the secretory pathway occurs sequentially - from organelle to organelle. Before reaching the final destination, the protein can visit several organelles (ER, different parts of the AG). The journey from the ER membrane to its destination can take tens, if not hundreds of minutes. During the transfer process, proteins can undergo significant modifications (primarily in AG). On final stages transport can occur in parallel - into the vacuole, periplasmic space or plasmalemma.
Finally, the two protein transport pathways differ in the mechanism by which molecules are transferred. For the cytosolic pathway, only a monomolecular protein transport mechanism is possible, in which each protein molecule individually crosses the membrane through the corresponding translocator. The secretory pathway is characterized by a vesicular mechanism for the transport of protein molecules, which is mediated by transport vesicles (vesicles). Vesicles are detached from one compartment, and certain molecules are captured from its cavity. The vesicles then merge with another compartment, delivering their contents into it. In vesicular transport, proteins do not cross any membranes; transport can only occur between topologically equivalent compartments. The vesicular transport mechanism is selectively controlled by special proteins that act as sorting signals. The protein enters the transport vesicle if its sorting signal binds to a receptor on the vesicle membrane. Currently, some sorting signals within proteins are known, while most of their complementary membrane receptors are not.
1.2. A plant cell is the result of a double symbiosis.
The existence strategy of higher plants is determined primarily by their two main properties - the phototrophic type of nutrition and the lack of active motility. These two properties left their mark on all levels of organization of the plant organism, right down to the cellular level.
In addition to the common ones eukaryotic cells characteristics, plant cells have a number of characteristics. The main ones:
presence of plastids; presence of vacuoles; presence of a rigid cell wall.
A diagram of the structure of a typical plant cell is shown in Fig. 1.3.
The presence of plastids is associated primarily with the phototrophic type of plant nutrition. Plastids, like mitochondria, have own genome. Thus, another feature of a plant cell is that it combines three relatively autonomous genetic systems: nuclear (chromosomal), mitochondrial and plastid. The presence of three genomes is a consequence of the symbiotic origin of plant cells. At the same time, the plant cell, unlike other eukaryotic cells, was formed from at least three initially independent forms:
1) a “host” organism, the genetic apparatus of which has moved to the nucleus;
2) a heterotrophic bacterium (similar to Rhodospirillum), which served as the predecessor of mitochondria;
3) an ancient bacterium with oxygenic photosynthesis (similar to the cyanobacterium synechocystis), which became the ancestor of plastids.
Long-term coevolution of symbionts led to a redistribution of functions between them and their genetic systems, with many mitochondrial and plastid DNA genes being moved into the nucleus.
In the metabolism of the body, the leading role belongs to proteins and nucleic acids. Protein substances form the basis of all vital structures of the cell; they are part of the cytoplasm. Proteins have unusually high reactivity. They are endowed with catalytic functions, i.e. they are enzymes, therefore proteins determine the direction, speed and close coordination and conjugation of all metabolic reactions.
Rice. 13 A. Scheme of protein synthesis in a eukaryotic cell.
Rice. 13 B. Scheme of protein synthesis in a prokaryotic cell.
The leading role of proteins in the phenomena of life is associated with the richness and diversity of their chemical functions, with the exceptional ability for various transformations and interactions with other simple and complex substances that make up the cytoplasm.
Nucleic acids are part of the most important organ of the cell - the nucleus, as well as the cytoplasm, ribosomes, mitochondria, etc. Nucleic acids play an important, primary role in heredity, body variability, and protein synthesis.
The process of protein synthesis is a very complex multi-step process. It is completed in special organelles - ribosomes. The cell contains a large number of ribosomes. For example, at coli there are about 20,000 of them.
How does protein synthesis occur in ribosomes?
Protein molecules are essentially polypeptide chains made up of individual amino acids. But amino acids are not active enough to combine with each other on their own. Therefore, before connecting with each other and forming a protein molecule, amino acids must be activated. This activation occurs under the action of special enzymes. Moreover, each amino acid has its own enzyme specifically tuned to it.
The energy source for this (as for many processes in the cell) is adenosine triphosphate (ATP).
As a result of activation, the amino acid becomes more labile and, under the action of the same enzyme, binds to t-RNA.
It is important that each amino acid corresponds to a strictly specific tRNA. She finds “her” amino acid and transfers it to the ribosome. Therefore, such RNA is called transport RNA.
Consequently, various activated amino acids enter the ribosome, connected to their tRNAs. The ribosome is like a conveyor for assembling a protein chain from various amino acids entering it (Fig. 13 A and B).
The question arises: what determines the order of binding of individual amino acids to each other? After all, it is this order that determines which protein will be synthesized in the ribosome, since its specificity depends on the order of amino acids in the protein. The cell contains more than 2000 specific proteins of different structure and properties.
It turns out that simultaneously with the t-RNA, on which its amino acid “sits,” the ribosome receives a “signal” from the DNA, which is contained in the nucleus. In accordance with this signal, this or that protein, this or that enzyme is synthesized in the ribosome (since enzymes are proteins).
The directing influence of DNA on protein synthesis is not carried out directly, but with the help of a special intermediary, that form of RNA, which is called messenger or messenger RNA (m-RNA or i-RNA).
Messenger RNA is synthesized in the nucleus under the influence of DNA, so its composition reflects the composition of DNA. The RNA molecule is like a cast of the DNA form.
The synthesized mRNA enters the ribosome and, as it were, conveys to this structure a plan - in what order the activated amino acids entering the ribosome should be connected to each other in order for a specific protein to be synthesized. Otherwise, the genetic information encoded in DNA is transferred to mRNA and then to protein.
The messenger RNA molecule enters the ribosome and, as it were, stitches it. That segment of it that is currently located in the ribosome, defined by a codon (triplet), interacts quite specifically with a triplet that matches it in structure (anticodon) in the transfer RNA, which brought the amino acid into the ribosome. Transfer RNA with its amino acid approaches a specific codon of the mRNA and connects with it; another t-RNA with a different amino acid is added to the next adjacent section of i-RNA, and so on, until the entire chain of i-RNA is read and until all the amino acids are reduced in the appropriate order, forming a protein molecule. And the tRNA, which delivered the amino acid to a certain part of the polypeptide chain, is freed from its amino acid and leaves the ribosome. Then, again in the cytoplasm, the desired amino acid can join it and again transfer it to the ribosome. In the process of protein synthesis, not one, but several ribosomes - polyribosomes - are involved simultaneously.
The main stages of the transfer of genetic information: synthesis on DNA as a matrix of i-RNA (transcription) and synthesis in ribosomes of a polypeptide chain according to the program contained in i-RNA (translation), are universal for all living beings. However, the temporal and spatial relationships of these processes differ among proeukaryotes.
In organisms that have a true nucleus (animals, plants), transcription and translation are strictly separated in space and time: the synthesis of various RNAs occurs in the nucleus, after which the RNA molecules must leave the nucleus, passing through the nuclear membrane (Fig. 13 A). The RNAs are then transported in the cytoplasm to the site of protein synthesis - ribosomes. Only after this comes the next stage - broadcasting.
In bacteria, the nuclear substance of which is not separated from the cytoplasm by a membrane, transcription and translation occur simultaneously (Fig. 13 B).
Modern diagrams illustrating the work of genes are based on logical analysis experimental data obtained using biochemical and genetic methods. The use of subtle electron microscopic methods allows one to literally see the work of the hereditary apparatus of the cell. IN lately Electron microscopic images were obtained, which show how on the bacterial DNA matrix, in those areas where molecules of RNA polymerase (an enzyme that catalyzes the transcription of DNA into RNA) are attached to the DNA, the synthesis of mRNA molecules occurs. The mRNA strands, located perpendicular to the linear DNA molecule, move along the matrix and increase in length. As the RNA strands lengthen, ribosomes join them, which, in turn, move along the RNA strand towards DNA and lead to protein synthesis.
From all that has been said, it follows that the place of synthesis of proteins and all enzymes in the cell are ribosomes. Figuratively speaking, these are like protein “factories”, like an assembly shop, where all the materials necessary for assembling the polypeptide chain of protein from amino acids are supplied. The nature of the synthesized protein depends on the structure of i-RNA, on the order of arrangement of nucleoids in it, and the structure of i-RNA reflects the structure of DNA, so that ultimately the specific structure of the protein, i.e., the order of arrangement of various amino acids in it, depends on the order location of nucleoids in DNA, on the structure of DNA.
The presented theory of protein biosynthesis is called the matrix theory. This theory is called matrix because nucleic acids play the role of matrices in which all the information regarding the sequence of amino acid residues in a protein molecule is recorded.
The creation of the matrix theory of protein biosynthesis and deciphering the amino acid code is the largest scientific achievement XX century, the most important step on the way to elucidating the molecular mechanism of heredity.
Mechanism of synthesis of non-protein substances
Nucleolus
This is a dense granule with a diameter of 1-3 microns, intensely stained with basic dyes. The main component of the nucleolus is a specialized region of chromosomes (loops), or the organizer of the nucleolus. Such regions are found in five chromosomes: 13th, 14th, 15th, 21st and 22nd; This is where numerous copies of genes encoding ribosomal RNAs are located.
In EM, 3 components are described in the nucleolus:
1. Fibrillar component- many thin (5-8 nm) filaments, with predominant localization in the inner part of the nucleolus. These are primary rRNA transcripts.
2. Granular component- this is a cluster of dense particles with a diameter of 10-20 nm; they correspond to the most mature precursors of ribosomal subunits.
3. Amorphous component– This is the zone where nucleolar organizers are located, a very pale colored zone. There are large DNA loops involved in the transcription of ribosomal RNA, as well as proteins that specifically bind to RNA. Granules and fibrils form nucleolar filament (nucleolonema), thickness 60-80 nm. Since the nucleolus is surrounded by chromatin, it is called perinuclear chromatin, and its part penetrating into the nucleolus is intranucleolar chromatin.
Cell Conveyor is the assembly of a secretory product on a living conveyor belt with the participation of various cellular organelles. In this case, the assembly process consists of a number of stages occurring in a certain sequence in areas of the cell that are quite far removed from the place of direct action nucleic acids that carry out genetic control.
The cellular conveyor belt for protein synthesis involves the usual sequence of processes outlined in the section describing the granular endoplasmic reticulum. Here it is appropriate to present the mechanism of synthesis of non-protein substances.
1. Transcription of DNA to form m-RNA
2. Formation of ribosomal RNA in the nucleolar zone
3. Assembly of the ribosome precursor in the nuclear zone
4. Entry of large and small ribosomal subunits into the cytoplasm
5. Synthesis of enzymes on free ribosomes for the biosynthesis of non-protein substances (carbohydrates and lipids)
6. The entry of enzymes into the hyaloplasm or smooth ER, where the synthesis of carbohydrates or lipids occurs
7. Entry of these substances into the Golgi complex, formation of a secretory granule with release from the cell or preservation of substances inside the cell
Thus, lipids and carbohydrates are synthesized in the cytoplasm and smooth ER and are packaged into CG with a (“minus membrane”) effect.
The enzymes involved in the biosynthesis of these lipids are integral membrane proteins, the catalytic sites of which face the cytosol. Synthesis occurs through several enzymatic reactions. New lipids diffuse freely in the plane of the bilayer and quickly mix with the lipids of the outer layer of the membrane. In addition, the enzyme flippase can move newly synthesized lipids into the inner layer of the membrane. This is how rapid mixing of glycerophospholipids occurs.
The contours of the cell, even at the light-optical level, do not appear even and smooth, and electron microscopy has made it possible to detect and describe various structures in the cell that reflect the nature of its functional specialization. The following structures are distinguished:
1. Microvilli – protrusion of cytoplasm covered with plasmalemma. The microvillus cytoskeleton is formed by a bundle of actin microfilaments, which are woven into the terminal network of the apical part of the cells (Fig. 5). Single microvilli are not visible at the light optical level. If there are a significant number of them (up to 2000-3000) in the apical part of the cell, even with light microscopy a “brush border” is distinguished.
2. Eyelashes – are located in the apical zone of the cell and have two parts (Fig. 6): a) outer - axoneme
b) internal – becal body
Axoneme consists of a complex of microtubules (9 + 1 pairs) and associated proteins. Microtubules are formed by the protein tubulin, and the handles are formed by the protein dynein - these proteins together form the tubulin-dynein chemomechanical transducer.
Basal body consists of 9 triplets of microtubules located at the base of the cilium and serves as a matrix for organizing the axoneme.
3. Basal labyrinth- These are deep invaginations of the basal plasmalemma with mitochondria lying between them. This is a mechanism for the active absorption of water, as well as ions against a concentration gradient.
1. Transport low molecular weight compounds carried out in three ways:
1. Simple diffusion
2. Facilitated diffusion
Active transport
Simple diffusion– low molecular weight hydrophobic organic compounds (fatty acids, urea) and neutral molecules (HO, CO, O). As the difference in concentration between the compartments separated by the membrane increases, the rate of diffusion also increases.
Facilitated diffusion– the substance passes through the membrane also in the direction of the concentration gradient, but with the help of a transport protein – translocases. These are integral proteins that have specificity for transported substances. These are, for example, anion channels (erythrocyte), K channels (plasmolemma of excited cells) and Ca channels (sarcoplasmic reticulum). Translocase for H O it is aquaporin.
Mechanism of action of translocase:
1. The presence of an open hydrophilic channel for substances of a certain size and charge.
2. The channel opens only when a specific ligand binds.
3. There is no channel as such, and the translocase molecule itself, having bound the ligand, rotates 180 in the plane of the membrane.
Active transport– this is transport using the same transport protein (translocases), but against a concentration gradient. This movement requires energy.
2. Transport of high-molecular compounds across membranes
The transition of particles through the plasmalemma always occurs in the composition membrane vesicle: 1. Endocytosis: A. pinocytosis, b. phagocytosis, c. receptor-mediated endocytosis.
Exocytosis: A. secretion, b. excretion, c. Recretion is the transfer of solid substances through a cell; phagocytosis and excretion are combined here.
- 1. OBJECTIVE OF THE LESSON: to study the structure of the interphase nucleus in fixed preparations. Consider the structural features of cell nuclei with different functional activities. The main components of the nucleus are: nuclear envelope (karyolemma), chromatin, nucleolus, nuclear juice. Under light microscopy, the nuclear envelope presents a clear line outlined from the side of the nucleus and cytoplasm. When considering the diagram of the ultramicroscopic structure of the nucleus, one should pay attention to the structural features of the karyolemma and the connection of its membranes with the endoplasmic reticulum of the cytoplasm. Understand the morphological characteristics of chromatin and its chemical composition. Chromatin in the nucleus can be in the form of clumps (condensed chromatin) or dispersed (dispersed chromatin). The different state of chromatin is an indicator of the biosynthetic activity of the cell. Cells that actively synthesize protein have a nucleus with dispersed chromatin and a well-developed nucleolus. In the nuclei of cells that do not synthesize protein, the chromatin is condensed, and the nucleoli are poorly visible.
- 2. Test questions: 1. Core. The concept of the interphase nucleus. Structural Components nuclei according to light and electron microscopy: nuclear envelope, chromatin, nucleolus, nuclear juice. The importance and functions of the nucleus in the life of the cell. 2. Nuclear-cytoplasmic ratios in cells with different levels of metabolism. 3. Structure of the nuclear envelope in SM and EM. Molecular organization and functional significance of the nuclear lamina. 4. Nuclear pore and nuclear pore complex. Participation in nuclear import and export of substances. 5. Chromatin of the interphase nucleus. Euchromatin and heterochromatin. Chromatin as an indicator of cell biosynthetic activity. 6. Molecular organization of DNA in chromosomes. Levels of chromatin folding. The role of histone proteins in ensuring the structure of chromatin and the implementation of genetic information. 7. Nucleolus. Structure of the nucleolus in SM and EM. Main components of the nucleolus. The role of the nucleolus in rRNA synthesis and ribosome formation. 8. Synthesis and transport of biopolymers in the cell. Cellular conveyor belt during protein synthesis. Morphological characteristics of a cell that synthesizes proteins. 9. Cellular conveyor during the synthesis of carbohydrates and lipids. Morphological characteristics of a cell that synthesizes carbohydrates and lipids.
- 3. Drug 1. Kernel structures. Ovary. Hematoxylin-eosin staining. Under low magnification, make a general overview of the microspecimen, find the growing follicle with the egg. Under high magnification, find a large round cell—an egg—and examine the structure of the nucleus. Pay attention to the nuclear envelope, nucleolus, and chromatin state. Draw an egg cell and label the structures of the interphase nucleus. Study the electron diffraction pattern of the nucleus. Draw the structure of the karyolemma and nuclear pore complex.
- 4. Preparation 1. Kernel structures. Ovary. Egg. Hematoxylin-eosin staining
- 5. Specimen 2. Pancreas. Hematoxylin-eosin staining. A cell that synthesizes protein. Under low magnification, make a general overview of the microscopic specimen and locate the exocrine part of the pancreas. Under high magnification, examine one cell, paying attention to the presence of a nucleolus and euchromatin in the nucleus, note the basophilia of the cytoplasm in the basal part of the cell and oxyphilia in the apical part.
- 6. Specimen 2. Pancreas. Hematoxylin-eosin staining. Cells that synthesize proteins
- 7. Preparation 3. Liver. Glycogen in liver cells. CHIC reaction. A cell that synthesizes carbohydrates. Under low magnification, make a general overview of the microslide and find a group of hepatocytes. Under high magnification, examine red-violet glycogen clumps in the cytoplasm of the hepatocyte.
- 8. Preparation 3. Liver. Glycogen in liver cells. CHIC reaction. A cell that synthesizes carbohydrates.
- 9. Specimen 4. Lipid inclusions in liver cells. Staining with osmic acid. Cell that synthesizes lipids. Under low magnification, make a general overview of the microslide and find a group of hepatocytes. Under high magnification, examine the cytoplasm of the hepatocyte, paying attention to lipid droplets colored black.
- 10. Specimen 4. Lipid inclusions in liver cells. Staining with osmic acid. Cells that synthesize lipids.