In all organisms (with the exception of some viruses), the implementation of genetic material occurs according to the DNA-RNA-protein system. At the first stage, information is rewritten (transcribed) from one nucleic acid to another. The proteins that regulate this process are called transcription factors.
What is transcription
Transcription is the biosynthesis of an RNA molecule based on a DNA template. This is possible due to the complementarity of certain nitrogenous bases that make up nucleic acids. Synthesis is carried out by specialized enzymes - RNA polymerases and is controlled by many regulatory proteins.
The whole genome is not transcribed at once, but only a certain part of it, called transcripton. The latter includes a promoter (the site of attachment of RNA polymerase) and a terminator (a sequence that activates the completion of the synthesis).
Prokaryotic transcripton is an operon consisting of several structural genes (cistrons). Based on it, polycistronic RNA is synthesized,containing information about the amino acid sequence of a group of functionally related proteins. Eukaryotic transcripton contains only one gene.
The biological role of the transcription process is the formation of template RNA sequences, on the basis of which protein synthesis (translation) is carried out in ribosomes.
RNA synthesis in prokaryotes and eukaryotes
The RNA synthesis scheme is the same for all organisms and includes 3 stages:
- Initiation - attachment of the polymerase to the promoter, activation of the process.
- Elongation - extension of the nucleotide chain in the direction from 3' to 5' end with the closure of phosphodiester bonds between nitrogenous bases, which are selected complementary to DNA monomers.
- Termination is the completion of the synthesis process.
In prokaryotes, all types of RNA are transcribed by one RNA polymerase, consisting of five protomers (β, β', ω and two α subunits), which together form a core-enzyme capable of increasing the chain of ribonucleotides. There is also an additional unit σ, without which the attachment of the polymerase to the promoter is impossible. The complex of the core and the sigma factor is called a holoenzyme.
Despite the fact that the σ subunit is not always associated with the core, it is considered part of the RNA polymerase. In the dissociated state, sigma is not able to bind to the promoter, only as part of the holoenzyme. After completion of initiation, this protomer separates from the core, being replaced by an elongation factor.
Featureprokaryotes is a combination of translation and transcription processes. Ribosomes immediately join the RNA that begins to be synthesized and build an amino acid chain. Transcription stops due to the formation of a hairpin structure in the terminator region. At this stage, the DNA-polymerase-RNA complex breaks down.
In eukaryotic cells, transcription is carried out by three enzymes:
- RNA polymerase l – synthesizes 28S and 18S-ribosomal RNA.
- RNA polymerase ll – transcribes genes encoding proteins and small nuclear RNAs.
- RNA polymerase lll - responsible for the synthesis of tRNA and 5S rRNA (small subunit of ribosomes).
None of these enzymes is capable of initiating transcription without the participation of specific proteins that provide interaction with the promoter. The essence of the process is the same as in prokaryotes, but each stage is much more complicated with the participation of a larger number of functional and regulatory elements, including chromatin-modifying ones. At the initiation stage alone, about a hundred proteins are involved, including a number of transcription factors, while in bacteria, one sigma subunit is enough to bind to the promoter and sometimes the help of an activator is needed.
The most important contribution of the biological role of transcription in the biosynthesis of various types of proteins determines the need for a strict system for controlling gene reading.
Transcriptional regulation
In no cell is the genetic material realized in full: only part of the genes is transcribed, while the rest are inactive. This is possible thanks to the complexregulatory mechanisms that determine from which DNA segments and in what quantity RNA sequences will be synthesized.
In unicellular organisms, the differential activity of genes has an adaptive value, while in multicellular organisms it also determines the processes of embryogenesis and ontogenesis, when different types of tissues are formed on the basis of one genome.
Gene expression is controlled at several levels. The most important step is the regulation of transcription. The biological meaning of this mechanism is to maintain the required amount of various proteins required by a cell or organism at a particular moment of existence.
There is an adjustment of biosynthesis at other levels, such as processing, translation and transport of RNA from the nucleus to the cytoplasm (the latter is absent in prokaryotes). When positively regulated, these systems are responsible for the production of a protein based on the activated gene, which is the biological meaning of transcription. However, at any stage the chain can be suspended. Some regulatory features in eukaryotes (alternative promoters, splicing, modification of polyadenellation sites) lead to the appearance of different variants of protein molecules based on the same DNA sequence.
Since the formation of RNA is the first step in the decoding of genetic information on the way to protein biosynthesis, the biological role of the transcription process in modifying the cell phenotype is much more significant than the regulation of processing or translation.
Determination of the activity of specific genes as inin both prokaryotes and eukaryotes, it occurs at the stage of initiation with the help of specific switches, which include regulatory regions of DNA and transcription factors (TFs). The operation of such switches is not autonomous, but is under the strict control of other cellular systems. There are also mechanisms of non-specific regulation of RNA synthesis, which ensure the normal passage of initiation, elongation and termination.
The concept of transcription factors
Unlike the regulatory elements of the genome, transcription factors are chemically proteins. By binding to specific regions of DNA, they can activate, inhibit, speed up or slow down the transcription process.
Depending on the effect produced, the transcription factors of prokaryotes and eukaryotes can be divided into two groups: activators (initiate or increase the intensity of RNA synthesis) and repressors (suppress or inhibit the process). Currently, more than 2000 TFs have been found in various organisms.
Transcriptional regulation in prokaryotes
In prokaryotes, the control of RNA synthesis occurs mainly at the initiation stage due to the interaction of TF with a specific region of the transcripton - an operator that is located next to the promoter (sometimes intersecting with it) and, in fact, is a landing site for the regulatory protein (activator or repressor). Bacteria are characterized by another way of differential control of genes - the synthesis of alternative σ-subunits intended for different groups of promoters.
Partly operon expressioncan be regulated at the stages of elongation and termination, but not due to DNA-binding TFs, but due to proteins interacting with RNA polymerase. These include Gre proteins and the anti-terminator factors Nus and RfaH.
The elongation and termination of transcription in prokaryotes is influenced in a certain way by the parallel protein synthesis. In eukaryotes, both these processes themselves and the transcription and translation factors are spatially separated, which means that they are not functionally related.
Activators and repressors
Prokaryotes have two mechanisms of transcription regulation at the initiation stage:
- positive - carried out by activator proteins;
- negative - controlled by repressors.
When the factor is positively regulated, the attachment of the factor to the operator activates the gene, and when it is negative, on the contrary, it turns it off. The ability of a regulatory protein to bind to DNA depends on the attachment of a ligand. The role of the latter is usually played by low molecular weight cellular metabolites, which in this case act as coactivators and corepressors.
The mechanism of action of the repressor is based on the overlap of promoter and operator regions. In operons with this structure, the attachment of a protein factor to DNA closes part of the landing site for RNA polymerase, preventing the latter from initiating transcription.
Activators work on weak, low functionality promoters that are poorly recognized by RNA polymerases or are difficult to melt (separate helix strandsDNA required to initiate transcription). By joining the operator, the protein factor interacts with the polymerase, significantly increasing the probability of initiation. Activators are able to increase the intensity of transcription by 1000 times.
Some prokaryotic TFs can act as both activators and repressors depending on the location of the operator in relation to the promoter: if these regions overlap, the factor inhibits transcription, otherwise it triggers.
| Ligand function with respect to the factor | Ligand state | Negative regulation | Positive Regulation |
| Provides separation from DNA | Joining | Removal of the repressor protein, activation of the gene | Removal of activator protein, gene shutdown |
| Adds factor to DNA | Delete | Repressor removal, transcription inclusion | Remove activator, turn off transcription |
Negative regulation can be considered on the example of the tryptophan operon of the bacterium E. coli, which is characterized by the location of the operator within the promoter sequence. The repressor protein is activated by the attachment of two tryptophan molecules, which change the angle of the DNA-binding domain so that it can enter the major groove of the double helix. At a low concentration of tryptophan, the repressor loses its ligand and becomes inactive again. In other words, the frequency of transcription initiationinversely proportional to the amount of metabolite.
Some bacterial operons (for example, lactose) combine positive and negative regulatory mechanisms. Such a system is necessary when one signal is not enough for rational control of expression. Thus, the lactose operon encodes enzymes that transport into the cell and then break down lactose, an alternative energy source that is less profitable than glucose. Therefore, only at a low concentration of the latter, the CAP protein binds to DNA and starts transcription. However, this is advisable only in the presence of lactose, the absence of which leads to the activation of the Lac repressor, which blocks the access of the polymerase to the promoter even in the presence of a functional form of the activator protein.
Due to the operon structure in bacteria, several genes are controlled by one regulatory region and 1-2 TFs, while in eukaryotes, a single gene has a large number of regulatory elements, each of which is dependent on many other factors. This complexity corresponds to the high level of organization of eukaryotes, and especially multicellular organisms.
Regulation of mRNA synthesis in eukaryotes
The control of eukaryotic gene expression is determined by the combined action of two elements: protein transcription facts (TF) and regulatory DNA sequences that can be located next to the promoter, much higher than it, in introns or after the gene (meaning the coding region, and not a gene in its full meaning).
Some areas act as switches, others don't interactdirectly with TF, but give the DNA molecule the flexibility necessary for the formation of a loop-like structure that accompanies the process of transcriptional activation. Such regions are called spacers. All regulatory sequences together with the promoter make up the gene control region.
It is worth noting that the action of the transcription factors themselves is only part of a complex multi-level regulation of genetic expression, in which a huge number of elements add up to the resulting vector, which determines whether RNA will eventually be synthesized from a particular region of the genome.
An additional factor in the control of transcription in the nuclear cell is a change in the structure of chromatin. Here, both total regulation (provided by the distribution of heterochromatin and euchromatin regions) and local regulation associated with a specific gene are present. For polymerase to work, all levels of DNA compaction, including the nucleosome, must be eliminated.
The diversity of transcription factors in eukaryotes is associated with a large number of regulators, which include amplifiers, silencers (enhancers and silencers), as well as adapter elements and insulators. These regions can be located both near and at a considerable distance from the gene (up to 50 thousand bp).
Enhancers, silencers and adapter elements
Enhancers are short sequential DNA capable of triggering transcription when interacting with a regulatory protein. Approximation of the amplifier to the promoter region of the geneis carried out due to the formation of a loop-like structure of DNA. Binding of an activator to an enhancer either stimulates the assembly of the initiation complex or helps the polymerase proceed to elongation.
The enhancer has a complex structure and consists of several module sites, each of which has its own regulatory protein.
Silencers are DNA regions that suppress or completely exclude the possibility of transcription. The mechanism of operation of such a switch is still unknown. One of the hypothesized methods is the occupation of large regions of DNA by special proteins of the SIR group, which block access to initiation factors. In this case, all genes located within a few thousand base pairs from the silencer are turned off.
Adapter elements in combination with TFs that bind to them constitute a separate class of genetic switches that selectively respond to steroid hormones, cyclic AMP and glucocorticoids. This regulatory block is responsible for the cell's response to heat shock, exposure to metals and certain chemical compounds.
Among the DNA control regions, another type of elements is distinguished - insulators. These are specific sequences that prevent transcription factors from affecting distant genes. The mechanism of action of insulators has not yet been elucidated.
Eukaryotic transcription factors
If transcription factors in bacteria have only a regulatory function, then in nuclear cells there is a whole group of TFs that provide background initiation, but at the same time directly depend on binding toDNA regulatory proteins. The number and variety of the latter in eukaryotes is enormous. Thus, in the human body, the proportion of sequences encoding protein transcription factors is about 10% of the genome.
To date, eukaryotic TFs are not well understood, as are the mechanisms of operation of genetic switches, the structure of which is much more complicated than the models of positive and negative regulation in bacteria. Unlike the latter, the activity of nuclear cell transcription factors is affected not by one or two, but by dozens and even hundreds of signals that can mutually reinforce, weaken or exclude each other.
On the one hand, activation of a particular gene requires a whole group of transcription factors, but on the other hand, one regulatory protein may be enough to trigger the expression of several genes by the cascade mechanism. This whole system is a complex computer that processes signals from different sources (both external and internal) and adds their effects to the final result with a plus or minus sign.
Regulatory transcription factors in eukaryotes (activators and repressors) do not interact with the operator, as in bacteria, but with control sites scattered over DNA and affect initiation through intermediaries, which can be mediator proteins, factors of the initiation complex and enzymes that change the structure of chromatin.
With the exception of some TFs included in the pre-initiation complex, all transcription factors have a DNA-binding domain that distinguishesthem from numerous other proteins that ensure the normal passage of transcription or act as intermediaries in its regulation.
Recent studies have shown that eukaryotic TFs can affect not only the initiation but also the elongation of transcription.
Variety and classification
In eukaryotes, there are 2 groups of protein transcription factors: basal (otherwise called general or main) and regulatory. The former are responsible for the recognition of promoters and the creation of the pre-initiation complex. Needed to start transcription. This group includes several dozen proteins that are always present in the cell and do not affect the differential expression of genes.
The complex of basal transcription factors is a tool similar in function to the sigma subunit in bacteria, only more complex and suitable for all types of promoters.
Factors of another type affect transcription through interaction with regulatory DNA sequences. Since these enzymes are gene-specific, there are a huge number of them. By binding to regions of specific genes, they control the secretion of certain proteins.
Classification of transcription factors in eukaryotes is based on three principles:
- mechanism of action;
- functioning conditions;
- structure of the DNA-binding domain.
According to the first feature, there are 2 classes of factors: basal (interact with the promoter) and binding to upstream regions (regulatory regions located upstream of the gene). This kindclassification essentially corresponds to the functional division of TF into general and specific. Upstream factors are divided into 2 groups depending on the need for additional activation.
According to the features of functioning, constitutive TFs are distinguished (always present in any cell) and inducible (not characteristic of all cell types and may require certain activation mechanisms). Factors of the second group, in turn, are divided into cell-specific (participate in ontogeny, are characterized by strict expression control, but do not require activation) and signal-dependent. The latter are differentiated according to the type and mode of action of the activating signal.
The structural classification of protein transcription factors is very extensive and includes 6 superclasses, which include many classes and families.
Operation principle
The functioning of basal factors is a cascade assembly of various subunits with the formation of an initiation complex and activation of transcription. In fact, this process is the final step in the action of the activator protein.
Specific factors can regulate transcription in two steps:
- assembly of the initiation complex;
- transition to productive elongation.
In the first case, the work of specific TFs is reduced to the primary rearrangement of chromatin, as well as the recruitment, orientation and modification of the mediator, polymerase and basal factors on the promoter, which leads to the activation of transcription. The main element of signal transmission is the mediator - a complex of 24 subunits acting inas an intermediary between the regulatory protein and RNA polymerase. The sequence of interactions is individual for each gene and its corresponding factor.
Regulation of elongation is carried out due to the interaction of the factor with the P-Tef-b protein, which helps RNA polymerase overcome the pause associated with the promoter.
Functional structures of TF
Transcription factors have a modular structure and perform their work through three functional domains:
- DNA-binding (DBD) - needed for recognition and interaction with the regulatory region of the gene.
- Trans-activating (TAD) – allows interaction with other regulatory proteins, including transcription factors.
- Signal-Recognizing (SSD) - required for the perception and transmission of regulatory signals.
In turn, the DNA-binding domain has many types. The main motives in its structure include:
- "zinc fingers";
- homeodomain;
- "β"-layers;
- loops;
- "leucine lightning";
- spiral-loop-spiral;
- spiral-turn-spiral.
Thanks to this domain, the transcription factor "reads" the nucleotide sequence of DNA in the form of a pattern on the surface of the double helix. Due to this, specific recognition of certain regulatory elements is possible.
The interaction of motifs with the DNA helix is based on the exact correspondence between the surfaces of thesemolecules.
Regulation and synthesis of TF
There are several ways to regulate the influence of transcription factors on transcription. These include:
- activation - a change in the functionality of the factor in relation to DNA due to phosphorylation, ligand attachment or interaction with other regulatory proteins (including TF);
- translocation - transport of a factor from the cytoplasm to the nucleus;
- availability of the binding site - depends on the degree of chromatin condensation (in the state of heterochromatin, DNA is not available for TF);
- a complex of mechanisms that are also characteristic of other proteins (regulation of all processes from transcription to post-translational modification and intracellular localization).
The last method determines the quantitative and qualitative composition of transcription factors in each cell. Some TFs are able to regulate their synthesis according to the classical feedback type, when its own product becomes an inhibitor of the reaction. In this case, a certain concentration of the factor stops the transcription of the gene encoding it.
General transcription factors
These factors are necessary to start the transcription of any genes and are designated in the nomenclature as TFl, TFll and TFlll depending on the type of RNA polymerase with which they interact. Each factor consists of several subunits.
Basal TFs perform three main functions:
- correct location of RNA polymerase on the promoter;
- unwinding of DNA chains in the region of the start of transcription;
- liberation of polymerase frompromoter at the moment of transition to elongation;
Certain subunits of basal transcription factors bind to promoter regulatory elements. The most important is the TATA box (not characteristic of all genes), located at a distance of "-35" nucleotides from the point of initiation. Other binding sites include the INR, BRE and DPE sequences. Some TFs do not directly contact DNA.
The group of major transcription factors of RNA polymerase ll includes TFllD, TFllB, TFllF, TFllE and TFllH. The Latin letter at the end of the designation indicates the order of detection of these proteins. Thus, the factor TFlllA, which belongs to the lll RNA polymerase, was the first to be isolated.
| Name | Number of protein subunits | Function |
| TFllD | 16 (TBP +15 TAFs) | TBP binds to the TATA box and TAFs recognize other promoter sequences |
| TFllB | 1 | Recognizes BRE element, accurately orients polymerase at initiation site |
| TFllF | 3 | Stabilizes polymerase interaction with TBP and TFllB, facilitates attachment of TFllE and TFllH |
| TFllE | 2 | Connects and adjusts TFllH |
| TFllH | 10 | Separates DNA chains at the point of initiation, frees the RNA-synthesizing enzyme from the promoter and major transcription factors (biochemistryprocess is based on phosphorylation of the Cer5-C-terminal domain of RNA polymerase) |
Assembly of basal TF occurs only with the assistance of an activator, a mediator and chromatin-modifying proteins.
Specific TF
Through the control of genetic expression, these transcription factors regulate the biosynthetic processes of both individual cells and the whole organism, from embryogenesis to fine phenotypic adaptation to changing environmental conditions. The sphere of influence of the TF includes 3 main blocks:
- development (embryo- and ontogeny);
- cell cycle;
- response to external signals.
A special group of transcription factors regulates the morphological differentiation of the embryo. This protein set is encoded by a special 180 bp consensus sequence called the homeobox.
In order to determine which gene should be transcribed, the regulatory protein must "find" and bind to a specific DNA site that acts as a genetic switch (enhancer, silencer, etc.). Each such sequence corresponds to one or more related transcription factors that recognize the desired site due to the coincidence of the chemical conformations of a particular outer segment of the helix and the DNA-binding domain (key-lock principle). For recognition, a region of the primary structure of DNA called the major groove is used.
After binding to DNA actionactivator protein triggers a series of successive steps leading to the assembly of the preinitiator complex. The generalized scheme of this process is as follows:
- Activator binding to chromatin in the promoter region, recruitment of ATP-dependent rearrangement complexes.
- Chromatin rearrangement, activation of histone-modifying proteins.
- Covalent modification of histones, attraction of other activator proteins.
- Binding additional activating proteins to the regulatory region of the gene.
- Involvement of a mediator and general TF.
- Assembly of the pre-initiation complex on the promoter.
- Influence of other activator proteins, rearrangement of subunits of the pre-initiation complex.
- Start transcription.
The order of these events may vary from gene to gene.
To such a large number of activation mechanisms there corresponds an equally wide range of repression methods. That is, by inhibiting one of the stages on the way to initiation, the regulatory protein can reduce its effectiveness or completely block it. Most often, the repressor activates several mechanisms at once, guaranteeing the absence of transcription.
Coordinated control of genes
Despite the fact that each transcripton has its own regulatory system, eukaryotes have a mechanism that allows, like bacteria, to start or stop groups of genes aimed at performing a specific task. This is achieved by a transcription determining factor that completes the combinationsother regulatory elements necessary for maximum activation or suppression of the gene.
In transcriptons subject to such regulation, the interaction of different components leads to the same protein, which acts as the resulting vector. Therefore, the activation of such a factor affects several genes at once. The system works on the principle of a cascade.
The scheme of coordinated control can be considered on the example of ontogenetic differentiation of skeletal muscle cells, the precursors of which are myoblasts.
Transcription of genes encoding the synthesis of proteins characteristic of a mature muscle cell is triggered by any of four myogenic factors: MyoD, Myf5, MyoG and Mrf4. These proteins activate the synthesis of themselves and each other, and also include the genes for the additional transcription factor Mef2 and structural muscle proteins. Mef2 is involved in the regulation of further differentiation of myoblasts, while simultaneously maintaining the concentration of myogenic proteins through a positive feedback mechanism.
