molecular biologists have uncovered the role of prokaryotic proteins of the Argonauts and found the exact mechanism of their work. It turned out that these proteins enhance the antiviral protection of cells, strengthening the impact of the recBCD system. In addition, the researchers directly demonstrated the existence of DNA-interference, which in the future can be used to study the architecture of genomes. Article published in Nature.
At the end of XX century in the course of an ordinary experiment in genetic modification had made a fundamental discovery. Scientists have tried to enhance the color of Petunia, a plant infecting viral particles, which contained an additional pigment gene. But instead strengthen the color she was missing. Later it turned out that this is the fault of RNA interference, and after some time were discovered proteins Argonauts (Argonaute) — part of its mechanism. These proteins can be contacted with short fragments of double-stranded RNA, and then looking in the cage molecule with the same sequence and destroy them. Having received a “target designation” in the attack of the input material, proteins Argonauts also destroyed native genes of the pigment, because they were identical.
It turned out that Argonaute is 60 percent of the eukaryotes whose genome was sequenced. Eukaryotic proteins of the Argonauts has been well researched, but then it turned outthat they have, and in prokaryotes, where it is not clear exactly neither their purpose nor the mechanism. In the study of 2014, it has been suggested that their function — protection against viruses and mobile elements, and that in addition to RNA interference in prokaryotes there is interference DNA. However, right this show failed, in addition, still remained unclear, from where the Argonauts take the short DNA-guides.
Anton Kuzmenko from the Institute of molecular genetics and his colleagues conducted experiments in vivo with protein Argonauts of prokaryotes Clostridium butyricum. These bacteria are very expensive, I live in anaerobic conditions and work with them hard. Therefore, scientists using the plasmid transferred the gene responsible for the production of Argonaute in cells of bacteria Escherichia coli (Escherichia coli). Along the way, to the gene were added to the sequence encoding several residues of histidine, since the loop of this amino acid good contact with metal ions, which gives the easiest way to extract the protein from the lysate of bacteria.
After E. coli multiplied, its suspension is passed through a high pressure homogenizer, which is hydraulically destroys the cells of bacteria, after which Argonaute extracted by the method of cobalt chromatography. To the extracted proteins were added to the phenol-chloroform mixture, which they have destroyed and freed galouye molecules.
Getting a free DNA molecule, the authors have otsenival them by the method of Illumina/Solexa and saw that most of the guides come from the same places of the genome, those where there is a termination of replication (ending the duplication of DNA). This led scientists to the idea that the segments of molecules are produced in system, homologous recombination repair (recBCD) that is valid in this region of the genome and cuts the DNA until, until you see the special brake light hee website (chi-site). When it was discovered that godovye sequence just limited to Chi-sites, the hypothesis was confirmed, and researchers have speculated that recBCD proteins and the Argonauts Clostridium butyricum act in conjunction and protects cells from viruses and mobile elements. Since Chi-sites is only in its genome recombination system repair completely destroys alien and trimming pick up proteins-the Argonauts and cut DNA with the same sequence, reinforcing the action of recBCD.
To experimentally confirm the protective functions of Argonaute, the scientists were introduced into E. coli, various viruses. Cells containing such proteins to survive under the action of the virus, and if not, were significantly longer.