Function and diversity of RNAbiogen.chuvsu.ru/uch_2_biol/carantin/Lesson10engl.pdfmRNA (or messenger RNA) is single stranded molecules. Molecule the mRNA can encode one or more polypeptide

Function and diversity of RNA

All types of RNA are formed in the reaction matrix synthesis, in most cases, the matrix is one of the DNA strands. The synthesis of RNA on the matrix DNA this process is called transcription, which involves the enzyme RNA polymerase (transcriptase).

1. Information/mRNA – contains a few 100-1000 nucleotides. Is open chain, carries information about the structure of a protein from DNA to the ribosome.2. Heterogeneous nuclear RNAS (tn-RNA) – is a precursor and is converted to RNA and RNA in result of processing. Usually tn-RNA longer than I-RNA.3. Ribosomal RNA – part of ribosomes and performs a structural function and participates in the synthesis of the polypeptide chain, is 85% of the total RNA. Cells prokaryotes contain 3 types of R-RNA and eukaryotes - 4.4. Transfer RNA – transfers amino acids to site of protein synthesis on ribosomes, each molecule of t-RNA contains 80 nucleotides. Its specificity is determined by the structure of the anticodon is the site of connection with a particular triplet of RNA.5. The small RNA. Are encoded in the nucleus, but function in the nucleus (small nuclear - SN) and in the cytoplasm (small cytoplasmic - SC). in-pRNAare part of the RNP (ribonucleoprotein complexes) involved in polyadenylation and splicing.6. Small interfering RNA (siRNA) has the ability to "turn off" genes.

The primary transcripts generated in the process of RNA synthesis, are often exposed after modifications or processing. As a result of the processing is the maturation of RNA. Only Mature RNA is able to perform tasks assigned to them.

mRNA (or messenger RNA) is single stranded molecules. Moleculethe mRNA can encode one or more polypeptide chains.mRNA, encoding information about a single polypeptide chain,called monocistronic, two or more polycistronic.RNA prokaryotes short-lived. Averagelife expectancy – 2 minutes.the mRNA of prokaryotes in most cases are polycistronic,and only some of them – monocistronic. Polycistronic mRNAMagistralnye contain noncoding region, whichshare the sites that encode the polypeptide chain (cistron). Withside of the 5’ end of the mRNA is non-coding leaderthe sequence containing the consensusthe sequence of the Shine-Dalgarno.

This sequence of six nucleotides AGGAGG . Complementary sequence UCCUCC called anti sequence of the Shine-Dalgarno is located on the 3/ end of the 16S rRNA molecule and determines the correct orientation of the mRNA in the ribosome during translation. From the 3’-end is non-coding end sequence (trailer). Coding sequence starts with an initiating codon (usually Aug, but can also be GUG) and ends with one of the termination codons (UAA, UAG or UGA).

Between cistrons region have a size of from 1 to 40 and more nucleotides.Sometimes ministrone region may be missing. Moreover, the last nucleotide of one cistron may be the first nucleotide following cistron, for example: AGAP.the mRNA of eukaryotes are monocistronic and constitute 2 – 6 % of all RNA in the cell. In eukaryotes genes are almost always single and only genes of t - and R-RNAS in clusters resembling bacterial operons.

Rodger Kornberg

In 2006 he was awarded the Nobel prize in chemistryfor the study of DNA transcription in eukaryotesOf the 10 000 litres of accumulated yeast culture, which corresponded to 150 kg themselves of yeast, has been allocated 2 g of net RNA polymerase.Kornberg was able to crystallographic picture of the various States of transcription apparatus, to establish the molecular structure of RNA polymerases and other proteins involved in the synthesis of mRNA and to identify the mechanisms that regulate the process of transcription.

Transcribers structure of the complex of RNA polymerase II (blue spiral DNA, red - synthesized mRNA)

Transcription in eukaryotes• Transcription in eukaryotes occurs in the nucleus

The synthesis of RNA molecules starts with the promoters, and ends at the sites of termination.Eukaryotes have 3 RNA polymerases (excluding mitochondrial and chloroplast):RNA polymerase I - synthesizes nucleoli ribosomal RNA (18S and 28S rRNAexcept 5S);RNA polymerase II synthesizes mRNA and some sRNA;RNA polymerase III - synthesizes tRNA, sRNA, 5S rRNA.

Transcription in eukaryotes

ТАТА-boxcontaining

About 24% of human genes

ТАТА-box not containing

Participation of various transcription

factors

• At a distance of 27 to 30 BP from the cap site is a TATA motif, a genericversion of which can be thought of as TATA(A/T)A(A/T).• The position of the TATA element strictly defines the site of initiationof transcription, i.e. the 5'end of the transcript.• In case of damage or deletion of the TATA element results in a set ofRNA molecules with different 5'-ends.• Individual base substitutions in the TATA-element, can lead to sharpdecrease in the efficiency of transcription.

Promotors

Transcription in eukaryotesInitiationActivation of the promoter occurs via a large protein – NANNY-factor, binds to the TATA box.

• Joining the TATA factor facilitates theinteraction of the promoter with RNApolymerase.

• Initiating factors cause the conformation change in RNA polymerase and provide for the unwinding of about one turn of the DNA helix, i.e., the transcriptional fork is formed in which the matrix is available to initiate the synthesis of RNA chain.

• Once synthesized ribooligonucleotidefrom 8-10 nucleotide residues, σ-subunit separates from the RNA polymerase, but instead to the enzyme molecule joined by several elongation factors.

Transcription in eukaryotesElongation• The elongation factors increases the activity of RNA polymerase and

facilitate the divergence of DNA chains.• At the stage of elongation, region of transcription forks,

simultaneously, separated by about 18 nucleotide pairs of DNA.• The growing end of the RNA chain forms a temporary hybrid helix of

about 12 pairs of nucleotide residues, with the matrix DNA.• As you move the RNA polymerase according to the matrix in the

direction from 3'- to 5'- end ahead of her is the divergence, and behind - the restoration of the DNA double helix.

Transcription in eukaryotesTermination

• Completes RNA synthesis in strictly defined areas of the matrix sites of termination of transcription

• The unwinding of the DNA double helix in the region of the site of termination makes it affordable for a factor of termination.

• Factor termination facilitates the separation of the primary transcript (pre-mRNA), complementary to the matrix, and RNA polymerase from the matrix. RNA polymerase can join in the next cycle of transcription after joining the subunits σ.

Transcription factor – regulates transcription by interacting with specific DNA or stoichiometric interacting with another protein, which can form a specific DNA sequence the complex "protein-DNA".In the human genome detected more than 2,600 proteins having a DNA binding domain, and most of them are believed to be a transcription factors.About 10% of all genes in the genome encode transcription factors, the largest family of human proteins.

Transcription in eukaryotes

Transcription in eukaryotesTranscription factors, binds to DNA that can affect gene transcription through several mechanisms:In most studied to date cases of TF to stimulate the formation of a complex of preinitial on TATA-box - initiator element due to the interaction of trans - activating domains with components of the basal transcription complex (either directly or through coactivators/mediators ).Some transcription factors cause changes in the structure of chromatin , making it more accessible to RNA polymerases .Other transcription factors are accessory to create the optimal conformation of DNA to other transcription factors.

Transcription in eukaryotes

4. Known transcription factors that repress transcription due to the direct actions of its inhibitory domains , or breaking a joint operation of the complex of transcription factors within a regulatory region of a gene (promoter, enhancer).

5. Finally, there are transcription factors that do not contact DNA, but are combined in more complicated complexes via protein - protein interactions.

Processing messenger RNAPrimary mRNA transcripts before they are used during protein synthesis, are subjected to several covalent modifications. These modifications are necessary for the functioning of mRNA as a matrix.

• Modification of the 5'-end• Modification of the 3'-end• Splicing of primary mRNA transcripts• Alternative splicing

Processing messenger RNAModification of the 5'-end

• Modification of pre-mRNA starts at the stage of elongation. Whenthe length of the primary transcript is about 30 nucleotide residues,there is copying its 5'end.

• Performs copying guanylyltransferase. The enzyme hydrolyzesmacroergic bond in the molecule GTP and attaches nucleotidebinding the remainder of the 5'-phosphate group to the 5'-end of thesynthesized RNA fragment with the formation of 5', 5'-fosfodiesterazyconnection.

Processing messenger RNA

• Modified 5'-end ensures the initiation of translation, lengthen the lifetime of the mRNA, protecting it from the action of 5'-economies inthe cytoplasm.

• Copying necessary for initiation of protein synthesis as initiating thetriplets AUG, GUG are recognized by the ribosome, only if there is acap. The presence of the cap is also essential for spliceosomeproviding the removal of its introns are.

Modification of the 5'-end

Encoding, or streaming, and (m)RNA starts from the initiator codon (Aug) and ends with one of three termination codons. The consensus sequence of Kozak (as described by Marilyn Kozak in 1986) plays an important role in the initiation of translation in eukaryotes, consists of four or six nucleotides preceding startcode (SASS mostly) and one or two (GC) nucleotide immediately after starttoday. (Author of figure: TransControl from English Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=2296784)

In eukaryotes, there are two mechanisms of finding a ribosome start codon AUG: cap – dependent (scan) and cap – independent (internal initiation) through IRES elements (eng. Internal Ribosomal Entry Site) — phase mRNAs with strong secondary structure, allowing him to direct the ribosome to start AUG.

Modification of the 3'-end• The 3'End of most transcripts synthesized by RNA polymerase II is

subjected to modification• A special enzyme, POLYa polymerase is formed by the POLYa sequence

(POLYa-tail), which consists of 100-200 residues of adenosine.• Signal the beginning of polyadenylation is the sequence AAUAAA on

the growing RNA chain. The enzyme POLYa polymerase, showing ectonucleoside activity breaks the 3' bond postepenno after the appearance in the RNA chain of a specific sequence - AAUAAA-.

Modification of the 3'-endTo the 3'end at the break point, POLYa polymerase increases by-lia-the "tail", the Presence of a POLYa sequence at 3'-end facilitatesthe yield of mRNA from the nucleus slows down its hydrolysis in the cytoplasm.The enzymes performing the caching, and polyadenylation,selectively associated with RNA polymerase II, and in the absence ofpolymerase inactive. Polyadenylation necessary fortransport of most mRNAs in the cytoplasm and protect moleculesmRNA from rapid degradation. Devoid of poly(A)-phase moleculesmRNAs are rapidly destroyed in the cytoplasm of eukaryotic cellsribonuclease. The half-life of the mRNA of eukaryotes is muchmore than in prokaryotes, and ranges from several hours toa few days.

Processing messenger RNASplicing of primary mRNA transcripts

• Sequence of its introns are "cut" from the primary transcript; the ends of the exons are connected to each other.

• The genes of eukaryotes contain its introns are longer than exons, so a very long molecule of pre-mRNA (about 5000 nucleotides) after splicing become shorter cytoplasmic mRNA molecules.

Processing messenger RNASplicing of primary mRNA transcripts

Processing messenger RNASplicing of primary mRNA transcripts• The process of "cutting out" its introns are

takes place with the participation of small nuclear ribonucleoproteins (snRNP).In the first stage of the process snRNPcontact splicing sites.

• Then joining other snRNP.• When forming the structure of

spliceosome the 3'end of one exon is moving closer to the 5'- end of the next exon.

• Spliceosome catalyzes the cleavage reaction of 3',5'-fosfodiesterazy connection on the boundary of the exon with introna.

• The sequence of intron is removed and the two exon join.

• The formation of 3',5'-fosfodiesterazy connection between the two exons catalyze snRNA fragment (small nuclear RNA) involved in the structure of spliceosome.

Processing messenger RNAAlternative splicing of primary mRNA transcripts

The stability and life time of mRNASome mRNAs in their 3'-UTR parts ARE elements (AU-rich element), with high frequency of adenine (A) and uridine (U).The binding of several proteins from the family of RBP (RNA-binding proteins) ARE element induces the degradation of mRNA.Early response genes that respond to a broad range of external signals, including oncogenes and cytokines, have relatively short life time because of ARE elements in their mRNA.Another type of RBP proteins, including HuR binding to the ARE regulates mRNA stability through the obstacles of access to it endonuclease.

Transport mRNA• Mature mRNAs are recognized by the presence of the modifications

and leave the nucleus through nuclear pores• In the cytoplasm the mRNA forms nucleoprotein complexes

informality, which is transported to the ribosomes• Many mRNAs contain signals that determine their localization

Ribosomes. In prokaryotic ribosomes there are three kinds of rRNAand ribosomal protein 55-60. Education rRNA in prokaryotes occurs as a result of processing of the primary transcript, containing nucleotide sequences of all three 16S, 23S and 5S rRNA, and tRNA. Upon maturation of the primary transcript occurs the separation of individual rRNA and tRNA.

The complete prokaryotic ribosome has a coefficient of sedimentaryand 70S and dissociates into two subunits. Small ribosomalthe subunit has a rod-shaped shape with a few smallledges and consists of one RNA molecule. Large subunitlike a hemisphere with the three protruding tabs. At the Association in the complete 70S ribosome small subcastes rests at one end on one the lugs 50S particles, and the other in her groove. As part of the big the molecules are 2 RNA molecules.

In eukaryotic – 4 70-85 rRNA and ribosomal proteins. Full eukaryotic ribosome 80S ribosome dissociate into small and large subunits similar to those of prokaryotes, but most is not two, but 3 RNA molecules. In total, the composition of the eukaryotic ribosome comprises four RNA molecules of different lengths: 28S RNA contains 5,000 nucleotides, 18S RNA – 2000, 5.8 S RNA – 160, 5S RNA of 120 nucleotides. (M.M. Yusupov, G.Zh. Yusupova, A. Baucom, K. Lieberman, T.N. Earnest, J.H.D. Cate, H.F. Noller (2001) Science 292: 883-896 )

16S ribosomal RNA 23S ribosomal RNA

Of them 18S, 5.8 S and 28S rRNA are synthesized in the nucleolus by RNA polymerase I as a single precursor (45S), which then is modified (methylated, mainly remnants of the ribose) and cutting. 5S rRNA is synthesized by RNA polymerase III in another part of the genome and require no additional modifications. The 5S rRNA forms the ribosome. Some primitive eukaryotes in the composition of the primary transcript of the nucleotide sequence corresponding to the 26S RNA contain introns. As a result of the processing of the primary transcript are cut out individual not only rRNA, but also intron is removed by autoplaying. For the acquisition of the functional activity of RNA in most cases should be subjected to complicated after modifications. Almost all rRNA is in the form of a magnesium salt, which is essential for maintaining the structure. Upon removal of magnesium ions is subjected to ribosome dissociation into subunits. Synthesis of ribosomes in eukaryotes takes place in a special nuclear structure, the nucleolus.

The number of nucleoli in the cells is usually from 1 to 5, but it is not constant, for example, in germ cells the number of nucleoli may reach several hundred.The increase in the number of nucleoli is called amplification of the nucleoli. The number nucleoli depends on the "nucleolar organizers" that are localized in the secondary constrictions of chromosomes. The more the number of "nucleolar the organizers of" the more nucleoli. The number of nucleoli increases, according to the the ploidy of the nucleus. The number of nucleoli is less than the number of "nucleolar the organizers of the" as "nucleolar organizers" can merge."Nucleolar organizers" are polycistronic areas that contain many genes (Polyethene areas), i.e. ribosomal genes. In the nucleus there are nucleolus, are not related nucleolar organizers. Copies of them can either be included in the composition chromosomes, either to become free. In the structure of the nucleolus are distinguished:Globular centre, a fibrillar centre, dense fibrillar component (PFK), chromatin, protein net matrix. On the surface fibrillary the centre is an activation of transcriptional units – pair with the transcription factors and Recolorado I which starts the count the primary rRNA transcript. As you progress through the first RNA polymerase the vacated site of the transcription unit sits next RNA polymerase begins the synthesis of new rRNA.

Peptidyltransferase rRNA catalyzes the reaction, a result of which the formation of peptide bonds in the process of protein synthesis on ribosomes. Maturation of ribosomes ends with the formation of functional ribonucleoprotein (complex rRNA and proteins).The ribosomal proteins. First, ribosomal proteins are mainly to appeal to the solvent surface ribosomes, and between subcortically proteins are almost there.Second, most ribosomal proteins have globular conformation, however, some contain elongated irregular plots, which are ordered tertiary structure when binding to rRNA. It was also found proteinsthat do not have a globular shape, for example, protein S14.Thirdly, the main role of ribosomal proteins, apparently, is the role of "staples" that stabilize the structure of the rRNA. However, partribosomal proteins may play an independent role in the compositionribosomes.Fourth, the secondary structure of rRNA is composed almost entirely of elements previously known for small fragments of RNA.

Two filament rRNA helical elements connected by loops that oftenrepresent irregular areas of A-helices RNA, and interact with small grooves (similar packaging I intron hammerhead ribozyme). Often such interactions are stable introduction of adenine to the small groove of the helix or interactions of rRNA phosphate group of one helix with a small second groove, and interactions of the unpaired purines in two perpendicular helices RNA. Ribosomal proteins interact with rRNAindependently of other protein and bind only with specific sites of rRNA, Called the core or primary RNA binding/The ribosomal proteins. The remaining ribosomal proteins are also associated with rRNA, but the binding sites of these proteins are formed only after Binding of the core proteins. In current models of ribosomal subcastes widely used to determine the position of antibiotics in the ribosome, and predictions of their contacts with rRNA.

tRNA molecules consist of about 75 nucleotides and phosphorylated 5'-end. The first reason is usually guanine. On the S'end, there are always three grounds - PAS and end IT. The share of tRNA is about 10 –15 % of the total cellular RNA. In tRNA are modified (minor) nitrogenous base (pseudouridine, dihydrouridine, thymidine, 7-methylguanosine, inosine, etc.). Their proportion can reach up to 25 %. More than half of the tRNA bases form pairs inside chain on the principle of complementarity.

tRNA recognizes the appropriate codon in mRNA and transports the required amino acidto the growing polypeptide chain.The recognition of the codon in mRNA is carried out using three consecutivebases in tRNA, called the anticodon. Between the nitrogenous bases of the codon and anticodon are formed Watson-Crick hydrogen bonds, provided that the polynucleotide chains are antiparallel.

Areas via N-links formed pair bases are called stems, and single stranded portions loops. All known tRNA will form a "cloverleaf" with four stems (acceptor, D, by anticodon and T) and threeloops (D, anticodon and T). Some tRNA have additional loops and/or stems (e.g., variable loop phenylalaninol yeast tRNA). Each stemconsists of two antiparallel chains forming right-doublethe spiral, known as A-form RNA. This form contains 11 pairsgrounds for a revolution, the pitch of the helix is equal to 3.1 nm. The plane of the bases be about 20° with the normal to the axis of the double helix. A-form RNA close to the similar form of DNA. RNA is not able to change conformation and go to In-form, which is due to the presence of 2'-group in the ribose, which is not in the deoxyribose. Spatial (tertiary) structure of a tRNA molecule resembles the shape of the letter G."Bar" this letter forms the spiral of the acceptor and T-stems and antikodonovye and D-stems form a "foot". In each part contains approximately 10 base pairs.

Amino acid residue prisoedinyaetsya to Z'-end of tRNA molecules.The specificity of such a transfer system is provided that there is at least one tRNA for each amino acid. So, tRNA is indicated for RA tRNA Phe.It should be emphasized that f involved in transcription are targets for number of biologically active substances, in particular antibiotics and toxins. For example, the pale toadstool toxin - α-amanitin - blocks RNA polymerase II, eukaryotes, which leads to the cessation of synthesis of new mRNA molecules, and many vital important proteins. Thus, a secondary structure, which received the name clover leaf. It is isolated: a) digidropiridinove branch with up to 3 residues dihydrouridine; b) pseudouridylation branch containing the minor nitrogenous base

pseudouridine;c) antikodonovye branch in the center of which is the anticodon, which

is complementary to antiparallel direction to the codon of the mRNA; d) additional branch. The number of components of its nucleotides

varies from 3 to 20. In some tRNA this branch is missing; e) acceptor branch of the universal 3'end by the sequence CCA, that

serves as the acceptor amino acid residue. Which is attached to the 3’-or 2’-gidroksilnuyu group of the remainder of the ribose of the last nucleotide.

Each amino acid usually has a few to appropriate to her tRNA, called isoacceptor. Isoacceptor tRNA are antikodons and are used to read different codons of the mRNA corresponding to the same amino acid. Total the number of tRNA genes in different organisms varies greatly(in Escherichia coli there are about 70, the clawed frogs Xenopus laevisabout 7 000, the person – 1300). Each tRNA gene can be represented in the genome of dozens of copies. Almost all tRNA are synthesizedin the form of predecessors – longer molecules (pre-tRNA). In the result of processing is the removal of the nucleotide sequences on the flanks of pre-tRNA. From the 5’end of the fragment the nucleotide chain cleaves an enzyme called RNAasa.RNAasa is ribonucleoprotein, a catalytic function in which provides the RNA component of the protein also has structural role. In bacterial RNase P there is a plot, complementary, CCA site tRNA. Eukaryotic RNAasa learns other elements of precursor tRNA. With the 3’-end of pre-tRNA acts accoucheuse, shortening the RNA gradually, removing one nucleotide.

In the final stages of maturation of tRNA 3’-end of thepolynucleotides attaches the sequence CCA.In eukaryotes, the CCA sequence is not encoded in tRNA genes, it is added after it. In prokaryotes the primary a transcript can contain multiple sequences of tRNA, their processing involves cutting the individual tRNAmolecules. In the maturation process of tRNA modification also occursnitrogenous bases – which formed the minor reason: pseudouridine, dihydrouridine, thymidine, 7-methylguanosine, inosine, etc.Canonical sequences at the boundary of intron and exon, characteristic of the pre-mRNA, pre-tRNA are missing. At the same time the composition has its introns are sequences complementary the anticodon. Pairing of these sequences with the anticodon, seem and leads to the formation of structures, providing a flow splicing.

In recent years has been the role of short RNA (siRNA). It is clear that from the first glance small RNA, consisting of only several tens of nucleotides, it might seem the remains of their "big brothers". And despite the fact that the role of individual small RNA molecules in the process of turning RNA (splicing), as well as the packaging of strands of nucleic acids has been proven before, the true "hit" in the biology of small RNAS began only with the opening of its ability to suppress gene expression in animals.

• In a normally operating cell each gene performs its own, specific function. For example, is responsible for the production of a protein, mRNA, or interaction with other regulatory proteins.

• While talking about the normal expression (from the Latin. expressus - expressive, explicit) gene in the cell. If the quantity of the product of this gene (e.g., protein) is reduced, we speak of the decrease in the expression of this gene. The effect of "damping" expression of specific genes in small RNA is called RNA interference, and molecule, causing it, called siRNA (small interfering Ribonucleic Acids, small interfering ribonucleic acid). With the discovery of RNAi interference has become clear that this phenomenon may have great practical importance.

RNA INTERFERENCEThe Nobel prize for discoveries in medicine and physiology in 2006, he received in equal shares by American scientists Andrew fire (Andrew Z. Fire. A citizen of the United States. Professor of pathology and of genetics Medical school Stanford University, USA) and Craig Mello (Craig C. Mello. A citizen of the United States. Professor, Department of molecular medicine Medical school University of Massachusetts, USA) for "the discovery of RNA interference - suppression genes double-stranded RNA".Small interfering RNA (siRNA) has the ability to "turn off" genes, influencing the process of transfer instructions between DNA and the rest of the "machinery" of the cell.Has exclusive specificity against their gene targets. The main function of this mechanism is protection of genetic information. The mechanism associated with siRNA protects us both from sporadic mutations or from outside attacks on our hereditary information.

RNA interference (RNAi, RNA interference) is a specific degradation of RNA molecules with the formation of double-stranded RNA molecules (siRNA) and their selective degradation

In a class of small RNAS include molecules containing from 20 to 300 nucleotides. The effect of RNA interference the short answer from them is siRNA consisting of only 21-28 (in mammals from 21-23) nucleotides. A feature of these molecules is that they, unlike most other cellular RNA, consisting of only one chain of nucleotides, are two filamentous. The nucleotides on opposite strands (chains) siRNA mate with each other under the same laws of complementarity that form two filamentous chains of DNA in chromosomes. In addition, the edges of each circuit siRNA is always the two unpaired nucleotide. As siRNA appear in the cell? Obviously, the cell must exist a molecular mechanism that would ensure the synthesis of siRNA, their accumulation in the cell and would allow them to turn off genes. Scientists have managed to identify the enzyme system, which is largely similar to all multicellular and some unicellular organisms.

If the siRNA molecule according to one reason or another (for example, according to the will of the researcher) appears in the cell, it immediately "takes turn" a special cellular protein system, for which the emergence of siRNA is a signal for immediate action. In the first stage, the siRNA molecule associated proteins-enzymes helicase nuclease and form a complex RISC (RNA-induced silencing complex; silence - eng.silent, silenced; in the English language and the literature referred to as the process of "turning off" the gene). Helicase spins siRNA strands, causing them to diverge. One of these threads, which is attached to the enzyme nuclease, can now contact a complementary plot one filamentous mRNA, allowing the nuclease to cut it. The cut areas of the mRNA are exposed to other cellular RNase that have cut them into smaller pieces.

Thus, the main "specialty" of siRNA in cell is blocking those genes, which correspond to one of the chains within the siRNA. With siRNA, the cell can defend itself from viruses. The genome of some of these dangerous barbarians consists of DNA, some from the RNA, and, against the usual rules, the RNA of the virus may be one and donetchiny. The process of cutting foreign (viral) mRNA in this case occurs by activation of the enzyme complex RISC. However, for greater efficiency of plants and insects invented a way of strengthening the protective action of siRNA. Joining chain mRNA, siRNA can plot using a set of enzymes, called DICER, to first finish the second chain of mRNA and then cut it in different places, creating, thus, a variety of "secondary" siRNA. They, in turn, form the RISC and mRNA is carried out through every stage, which was discussed above, up to its complete destruction.

• Such "secondary" molecule-specific will be able to contact not only the site of viral mRNA, which was sent to the "primary" molecule, but also to other areas, which dramatically increases the efficiency of cellular protection.

• Thus, in plants and lower organisms RNAi animals are an important part of a kind of "intracellular immunity" that allows you to quickly recognize and destroy foreign RNA. In that case, if in a cage penetrated RNA -containing virus, such protection will not allow him to breed. However, if the virus contains DNA, siRNA system will prevent him to produce viral proteins (as required for this mRNA will be recognized and be cut), and using this strategy will slow down its spread through the body.

In mammals, unlike insects and plants, works and other protection system. When injected into Mature (differentiated) cell of a mammal a foreign RNA, the length of which is greater than 30 nucleotides, the cell begins the synthesis of interferon. The interferon binds to specific receptors on the cell surface, is capable of stimulating in the cell the whole group of genes. As a result, in the cell is synthesized by several types of enzymes that inhibit protein synthesis and break down viral RNA. In addition, interferon can act on the adjacent, not yet infected cells, thus blocking the possible spread of the virus.

As you can see, both systems are largely similar: they have a common goal and "methods" work. Even the name "interferon" and "(RNA) interference" come from a common root. But they have one very significant difference: if the interferon at the first sign of invasion just freezes the cells, not allowing (just in case) the production of many, including "innocent" proteins in the cell, the siRNA system is extremely selective: each siRNA will recognize and destroy only their specific mRNA. Replacement of only one nucleotide within the siRNA leads to a dramatic decrease in the interference effect.

This is the main advantage of siRNA: none of the blockers of genes known so far, has the exclusive specificity against their gene targets. However, as seen in the many dangerous viral diseases in humans, no immune, neither interferon protection is not omnipotent, so we have time to borrow somebody's best practices in the fight against viruses. Why not in plants or insects? Neither one nor the other do not possess adaptive immunity. To survive, plants have been forced to "invent" RNA interference, which is still successfully protects their cells from introducing viruses. There is a natural question: is it possible to apply the same approach in relation to the cells of animals and people?

The genome of any multicellular organism involves many elements that were brought in in the process of evolution from the outside, for example as a result of the embedding of the virus. Judge for yourself: all of the material contained in our chromosomes, accounted for 34% of the proportion of elements called LINEs and SINEs (respectively, Long and Short Interspersed Nuclear Elements), the functions of which we only know that they can at times able to replicate itself and move from one place to another chromosome; the DNA that came from retroviruses (8% of the genome) and transposons (3%) are also able to change their location in the genome. On the background of only 2 (two(!)) percent actually of the genes encoding our proteins of cell, seem so unimportant a detail as siRNA among the huge variety of their big "sisters".

LINEs, SINEs, remnants of viral DNA and transposons, for its ability to move called mobile or transposable elements of the genome, pose a significant risk to our chromosomes. "Strangers among us", they under certain circumstances can raise a rebellion and lead to intracellular chaos. Some of them - the remnants of a virus, or proto-oncogenes capable of when "on" can cause cancer; transposable elements, multiplying and moving, changing the structure of chromosomes that can lead to mutations. For example, a favorite object of genetic research is the fruit fly Drosophila, more than 80% of spontaneous mutations that arise specifically because of the "hooligan" behavior of its own mobile items. Their movement within the genome is so individual and unpredictable that the position of some of them can serve as a "molecular passport", just determining the identity of the host, which is already used in practice.

• System of intracellular siRNA performs a "surveillance" function of the behavior of mobile elements. On the model of the same C. elegans, for example, it has been shown that deactivation of the genes encoding some of the small RNAS, leads to activation of the movements of mobile elements in the chromosomes and, consequently, to increase the level of mutations.

• In addition, errors in the development of organs and tissues when you disable the genes encoding the siRNA system in experimental animals and its activity in immature cells indicate that the mechanism of RNA interference is actively involved in the regulation of the program "maturation" of the cells and, consequently, can play a key role in the formation of the whole organism.

• One of the expected normal functions of siRNA - tracking incorrectly processed copies of other types of RNA in the cell.There is evidence that in some cases siRNA apparently acts directly on DNA, altering chromatin structure and promoting long "silencing" some, and possibly activation of other genes.

The multifunctionality of RNA(1) Encoding function: programming protein synthesis of the linear sequences of polyribonucleotides.(2) the Replicative function: replication of genetic material via complementary sequences of polynucleotides.(3) Structural function (the formation of three-dimensional structures): self-folding of linear polyribonucleotides in a unique compact conformation.(4) the Function of recognizing specific ligands: specific spatial interactions with other macromolecules and small ligands.(5) Catalytic function: specific catalysis of chemical reactions by ribosoms.

CODING FUNCTIONS OF RNA

(1) RNA (and DNA!) can serve as a matrix for private playback through the complementary chains of RNA:1962-1964 – the discovery of self-reproduction polioviruses RNA in animal cells (D. Baltimore).1965-1966 – discovery-replication of RNA bacteriophages of the type of R17 and MS2 in bacterial cells (S. Spiegelman).(2) RNA can serve as a matrix for DNA synthesis:1970 – opening of the reverse transcription (H. M. Temin, D. Baltimore).

The catalytic function of RNA (ribozymes)

(1) The discovery of natural ribozymes:K. Kruger, P.J. Grabowski, A.J. Zaug, J. Sands, D.E. Gottschling and T.R. Cech (1982) Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequences of Tetrahymena. Cell 31: 147-157.C. Guerrier-Takada, K. Gardiner, T. Marsh, N. Pace and S. Altman (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35: 849-857.

(2) The creation of artificial ribozymes:D.L. Robertson and G.F. Joyce (1990) Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344: 467-468.Reviewed in: T.R. Cech and B.L. Golden (1999) Building a catalytic active site using only RNA. In The RNA World, 2nd Edition (Eds. R.F. Gesteland et al.), CSHL Press, New York.

Catalytic (natural and artificial)the functions of RNA (ribozymes)

• Hydrolysis fostering relations• Transesterification• Ligation and polymerization of nucleotides including the matrix DNA or RNA• Alkylation and aminoacridone nucleotides• Synthesis of amide (peptide) bonds between amino acids• Transpeptidase• Synthesis of carbon-carbon bonds

CATALYTIC (NATURAL AND ARTIFICIAL)RNA FUNCTIONS (RIBOZYMES)

• Hydrolysis of phosphoether bonds• Transesterification• Ligation and polymerization of nucleotides, including on a DNA or RNA template• Alkylation and aminoacylation of nucleotides• Synthesis of amide (peptide) bonds between amino acids• Transpeptidation• Synthesis of carbon-carbon bonds

The function of recognizing small ligands (substrates)

(1) E. Cundliffe (1986) Involvement of specific portions of ribosomal RNA in defined ribosomal functions: A study utilizing antibiotics. In Structure, Function, and Genetics of Ribosomes (Eds. B. Hardesty

and G. Kramer), p.p. 128-142. Springer-Verlag, New York."Still not a suspect the ability of RNA to recognize small molecules".

(2) the Discovery and development of aptamers. Methodology SELEX’a.А. Ellington and J. Szostak (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346: 818-822.C. Tuerk and L. Gold (1990) Systematic evolution of ligands by exponential enrichment. Science 249: 505-510.

The function of recognizing small ligands (substrates)

(3) Demonstration of specific interactions of natural RNA with small molecules.D. Fourmy, M.I. Recht, S.C. Blanchard and J.D. Puglisi (1996) Structure of the A site of E. coli 16S rRNA complexed with an aminoglycoside antibiotic. Science 274: 1367-1371.

Three-dimensional structures of complexes of RNA with aminoglycoside antibiotics.(a) Paromomycin-A-site RNA complex. (b) Tobramycin-RNA aptamerscomplex.Reproduced from J.D. Puglisi and J.R. Williamson (1999) In The RNA World, 2nd Edition (Eds. R.F. Gesteland et al.), CSHL Press, New York.

Thus, RNA is able to carry out all the basic functions characteristic of both DNA and proteins. Therefore, ensembles of RNA molecules with different, complementary functions can be self-sufficient as assimilating, metabolizing, replicating, and structural systems, i.e. to be the prototype of living systems.