07 October, 2011

The got immunity at bacteria can be connected with RNK-INTERFERENCE mechanisms

The device bacteriophage is rather uniform — relations between phages and bacteria are essentially more various. They represent the whole spectrum: from commensalism to terrible parasitism. And each type of relations is made out by special biochemical means. A photo from a site mansfield.osu.edu.

The European scientists have deciphered one of stages of work of immune system CRISPR at bacteria. Though this system and widespread among bacteria and archaea, its components in many cases appeared highly specific. Besides, the enzymes participating in registration of the immune answer, have not been connected in any way with other sites of a bacterial metabolism. Now scientists have shown that on one of necessary stages of work CRISPR of a bacterium use, first, universal enzyme RNase, and secondly, extended at eukaryotes the mechanism of preparation of the immune answer, similar to the RNK-INTERFERENCE. Thus, there was a possibility more substantially to interpret an origin and evolution of immunity at bacteria.
Scientists all thoroughly plunge in micro- or even nanozhizn, opening all new mechanisms of its functioning. Each time, considering discussed research, you do not cease to be surprised to completeness, variety and orderliness of the device of life on that inaccessible to ordinary perception, level. Meanwhile the problems facing organisms of any scale level, more or less the general — varies only toolkit.

Sometimes for performance of this or that problem the universal tooling which the most different cages own or which suit many problems is used. But strictly specific set for a specific target is more often used. At level of cellular problems as tools various metabolic ways and the enzymes connected with them are used. A finding of universal cellular tools always the big good luck. If those are, it is possible to argue on their origin and evolution, about importance of problems solved with their help and etc . If the tool specific and is aimed at concrete function at a concrete cage, about its origin it is difficult to conclude something defined.

The new research presented by the European biochemists from universities Umeo (Sweden), Vienna (Austria) and a Würzburg (Germany), is connected with discussion of such universal tools of a cage. Research is devoted decoding of work of CRISPR-system which urged to provide protection against an infection.

CRISPR (clustered regularly interspaced short palindromic repeats) are special sites genomes bacteria and archaea, occupied with protection from virus and plasmid attacks and immunity development. "Elements" already addressed to this theme (Bacteria see inherit the got immunity, 1/21/2010), is is therefore only short summarised base positions. The CRISPR-cartridge represents a site genome on which are located, first, short palindromic sequences, secondly, slices of sequence virus DNK/RNK, so-called spacers, thirdly — a series of the specific fibers-nukleaz, called "Cas-fibers" (CRISPR associated proteins). Spacers and Palindromes strictly alternate, and this chain, actually, and is called CRISPR.

At penetration of a virus into a cage its site DNK/RNK is copied and inserted in a bacterium gene. The insert of this new spacer occurs with the assistance of Cas-fibers. Descendants of such cage will bear in the genome given virus spacer. A site genome, bearing inserted spacer, will be normal to be read out and make finally short РНК, including palindrome and this spacer. If the virus again gets to a cage by means of received short RNK there will be a recognition alien complementary DNA fragment (or RNK), and the cage will destroy nucleotide a chain of a parasite. Sequence CRISPR is constantly completed on a measure infection by new viruses, and spacer's which long time were not used, mutate, go out of use and disappear from a chain. It some kind of the dynamical system of the got immunity stopping an infection at level of a transcription and, thus, providing stability of cages to virus infections.

The problem which is solved by system CRISPR, — is universal, and the scheme of its decision logically accurate, therefore, apparently, and tools for its performance should be more or less uniform at prokaryotes. But actually scientists cannot brag of decoding of base principles of work CRISPR yet. So, the lecture of Ireny Artamonovoj devoted CRISPR, has been named «Adaptive immunity prokaryotes, based on crispr-systems: it is a lot of questions and few answers». One of important questions is connected with phylogeny Cas-fibers. It is considered that they serve all operations and acquisitions new spacer's and their successful functioning. The number of Cas-fibers in the cartridge very much differs at different bacteria and archaea: it varies from 20 to 4. They can be similar on structure at different families of bacteria, and at other bacteria to differ even within one strain. It means that any of fibers Cas or their separate domains cannot apply for a rank of the universal tool of CRISPR-immunity. Probably, any universal tool also is not present, and bacteria each time invent something approaching a case. But also it is possible, it is necessary what to search in another way and in other place.

The European command investigated CRISPR system at bacterium Streptococcus ruogenes. At this bacterium two CRISP-complete sets, one of which is, apparently, in a non-working condition. The worker, judging by sequences spacers, is directed on protection from lysogenic (moderate) phages. In the working cartridge besides palindromes and spacer's there are 4 more genes of Cas-fibers. All of them are involved only in process of work CRISPR. Primary reading leads to occurrence of chains RNK from 511 nucleotide. He/she is the so-called predecessor crispr-RNK which is called pre-cr RNK . Then this long RNK is cut on the necessary fragments "palindrome-Spenser", so-called crРНК which, actually, and are engaged in recognition phage sequences. Differently, there is a maturing process — transformation primary transcript in working fragments cr RNK .

However, as it was found out, at reading pre-crRNK from the CRISPR-cartridge there is one more short RNK, before unknown participant CRISPR of immunity. Its sequence is located on an opposite chain of DNA a little ahead of the cartridge. This RNK during maturing too is cut definitely. Scientists named it literally «suprotiv-activated crRNK» (trans-activating CRISPR RNA or, shortly, tracrRNA). It also became the main heroine of their scientific plot.

  The basic feature found out tracrRNA is a presence of a site from 25 nucleotide, which complementary all palindromic to repetitions CRISPR. It means that tracrRNA contact repetitions, and this process what for is necessary for formation of workers crРНК. To confirm this hypothesis it was possible, having created bacteria, mutant in area tracrRNA. At such bacteria of maturing crRNK did not occur. If to such defective bacteria to add normal tracrRNA maturing process renewed, producing normal crRNK. It is obvious that tracrRNA, coupling with палиндромами, forms the two-chained fragments RNK which ends anyhow define borders future crRNK. 

The RNK-INTERFERENCE mechanism: two-chained RNK it is distinguished endonucleaseDicer and it is cut on short 25 nukleotidnye fragments siRNA. The antisemantic chain joins complex RISC, and they together find complementary mRNK. Identified thus mRNK it is cut on pieces and degrades. The scheme from a site nobelprize.org

And here, naturally, it is necessary to remember, how there is a process of the RNK-INTERFERENCE at eukaryotes. Two-chained virus RNK it is identified by enzyme Dicer, it is cut on fragments from 25 nucleotide's, the double chain of a fragment untwines on one-chained RNK. Then one of threads — antisemantic siRNK (siRNA) — is attached to complex RISC. This design finds alien matrix RNK with a site, complementary the leader siRNK. Pairing of two threads makes active the enzyme cutting alien matrix RNK on pieces. Enzyme Dicer identifies only two-chained RNK and though RNK viruses can consist and of one chain, all the same in their life cycle necessarily there is a two-chained phase.

In our CRISPR-system too it will be organised two-chained RNK from 24 nucleotide, but at the expense of imposing tracrRNA which is available at thrifty Streptococcus ruogenes. Then this two-chained complete set should be identified endonuclease. Enzyme Dicer at bacteria is not present — that serves as its bacterial analogue? It has appeared that its role carries out bacterial РНКaza III. One of Cas-fibers — fiber Csn1 helps it. It anchored tracrRNA on long pre-crRNK, allowing endonuclease to get into gear. RNKaza III it is rather conservative, has no specific communication with CRISPR-system and participates in many processes, in particular provides maturing ribosomal RNK. Here this enzyme has joined in defensive actions against phages. So lines of the universal tool, probably, it is necessary to search not among Cas-fibers, and analyzing common features RNK of an interference and CRISPR protection, and also among other tools for work with RNK.

The scheme of participation of necessary components in work CRISPR. At first the predecessor pre-crRNA (the top line is read out: black — repetitions, green — v); together with it are read out c an opposite thread tracrRNA. To a place of events it is tightened RNKaza III and Csn1. Csn1 promotes pairing tracrRNA with repetitions CRISPR, then RNKaza III learns short two-chained fragments and cuts in a concrete place a two-chained complex on fragments "repetition-spejser-repetition". At the second step of maturing working fragments crRNK from 39–42 nucleotide's are created; the mechanism of this second step while is unknown, therefore on the scheme there are questions. The scheme from discussed article in Nature

Thus, for successful preparation of immune protection of a bacterium are necessary tracrRNA, RNKaza III and Csn1. Two of them — tracrRNA and Csn1 — concern to CRISPR to system, and one — nonspecific enzyme for work with RNK. tracrRNA together with Csn1 were at five kinds of bacteria but while because of a lack of the data it is difficult to define their real prevalence among bacteria.

Source: Elitza Deltcheva, Krzysztof Chylinski, Cynthia M. Sharma, Karine Gonzales, Yanjie Chao, Zaid A. Pirzada, Maria R. Eckert, Jörg Vogel, Emmanuelle Charpentier. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III //Nature. 2011 V. 471, P. 602–607.

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