Ribo-T is a synthetic ribosome with a single RNA chain
Two ribosomal subunits were combined into a functional hybrid
Yulia Kondratenko, "Biomolecule"
Bioengineers have developed hybrid ribosomes, which instead of two long rRNA chains include a single molecule that ensures the "indivisibility" of the organelle. Such ribosomes can support the synthesis of all the proteins necessary for a bacterial cell, although their translation initiation is much slower than that of conventional ribosomes.
Connected by a single chain. The subunits of an ordinary ribosome are functionally bound by matrix RNA – temporarily. The subunits of the new synthetic ribosome Ribo-T are bound by a single ribosomal RNA – forever.
Drawing from the website imglop.com .Ribosomes – molecular machines for protein synthesis – are similar in all known organisms.
The ribosome always has a small subunit that binds to the matrix RNA (mRNA) molecule, as well as a large subunit that joins them later. The functions of the subunits differ: the task of the small one is to identify the appropriate RNA and the place on it from which to start protein synthesis, and the large subunit containing the catalytic center joins the entire structure when everything is ready – completing the creation of a working ribosome. Premature connection of the ribosome halves is so undesirable that there are even special protein factors in the cell that prevent this.
The fact that the ribosome consists of two separate halves is also important for the continuation of polypeptide synthesis. The rotations of the subunits relative to each other drag the mRNA through the active center, so that all its sections that need to be read get into it in a timely manner. Finally, the separation of the subunits that counted the entire coding part of the RNA chain marks the end of the synthesis of the protein molecule.
Nevertheless, bioengineers really wanted to get ribosomes in which the subunits would be combined into one molecule and, despite this, could carry out all stages of translation. The subunits of such "machines" would not mix with the pool of ordinary ribosomes of the cell, so they could be experimented on without affecting the usual processes of synthesis of cellular proteins. Such a separate set of ribosomes could be adjusted to the production of unusual peptides: for example, to rebuild the exit tunnel of the ribosome so that large molecules could leave it, or to modify its active center for specific reactions.
RNA molecules serve as scaffolds of ribosomal subunits. Therefore, it is possible to fasten the subunits together by obtaining a single extended ribosomal RNA (rRNA) molecule, which includes frameworks for both subunits. At the same time, the new RNA should differ as little as possible from the sum of the original ones, so that cellular error control systems do not recognize the new molecule as mistakenly synthesized or foreign. The easiest way would be to connect the ends of ribosomal RNAs with chains of nucleotides. But the ends of the rRNA of the large and small subunits of natural ribosomes are located far from each other. But the two ends of the RNA of the large subunit are brought together. Therefore, they can be sewn, and new ends can be made elsewhere. The resulting variants are called circular permutants of the large subunit RNA.
Scientists had to get a large collection of such mutants, because it was not known where it was better to make new ends of rRNA. By chance, one of the harmless breaks in the RNA of the large subunit of the 70S ribosome was located where the large subunit contacts the small part, which, according to past studies, treats modifications calmly. Therefore, the scientists decided to connect the RNA in this place. They also had to choose the correct length of the ribonuclein ligaments: they had to be not too short so that the connected halves of the ribosome could move relative to each other, but also not too long so that one of the free cellular large subunits could not attach to the small subunit at the start of protein synthesis. Experience has shown that the appropriate length of linkers is eight and nine nucleotides.
Combining two rRNAs to produce a single and indivisible ribosome Ribo-T. Above is the secondary structure of native rRNAs (on the left) and their hybrid. The natural ends of 16S and 23S rRNAs are located in the ribosome subunits in such a way that it is not convenient to stitch them (the ends of the RNA are marked as 3’ and 5’). Therefore, scientists had to connect the ends of the RNA of the large subunit (the place of crosslinking (C) of 23S rRNA is marked in green at the top right) and make new ends where it would be convenient to connect them to 16S rRNA. Short chains of RNA linkers T1 and T2 (marked in red) were used for crosslinking. Below are models of a conventional bacterial ribosome (left) and a synthetic Ribot-T. A drawing from Orelle et al.With the resulting hybrid ribosomes Ribo-T (from the English tethered – related), scientists supplied bacterial strains that do not develop conventional ribosomes.
It turned out that hybrids are quite capable of supporting the life of bacteria and synthesizing all the proteins necessary for them. However, the growth rate of bacteria carrying only hybrid ribosomes was two times less than that of bacteria with ordinary ribosomes. And even this speed was achieved only by cells with a mutation in the gene of one of the ribosomal proteins and impaired synthesis of the supposed transporter of magnesium and cobalt ions. As well as the growth rate, the rate of protein synthesis in bacteria carrying hybrid ribosomes turned out to be two times less than normal.Scientists decided to find out why hybrid ribosomes synthesize protein more slowly.
It turned out that ribosomes with bonded subunits slow down immediately after recognition of the start codon. That is, the main problem for them is the correct initiation of the broadcast. Scientists have found that this problem is not associated with the worst interaction of hybrid ribosomes with initiation factors, since their addition did not compensate for the initial inhibition. So the new version of ribosomes has yet to be studied and improved in more detail.
Hybrid ribosomes, though not very fast, were doing their job, so scientists decided to test their suitability to work as an independent population of ribosomes. Firstly, ribosomes of a particular population can be used to read mRNA with unusual starting signals. To do this, hybrid ribosomes were obtained with a modified site recognizing the Shine-Dalgarno sequence – the starting point for planting bacterial ribosomes on RNA. The RNA with the altered Shine-Dalgarno sequence was not recognized by ordinary cellular ribosomes, but its product was found in the cytoplasm of cells carrying specially modified hybrid ribosomes. This means that only hybrid ribosomes were engaged in protein synthesis on such a matrix, and the translation of RNA with specific signals can be studied without affecting the translation of the remaining mRNAs of the cell.
Scientists also received a modification of hybrid ribosomes dependent on an antibiotic. Such ribosomes will work if an antibiotic is added to the medium that blocks the normal ribosomes of the cell. An antibiotic in such a system serves as a switch that determines which set of ribosomes will be active. So the cells can be maintained either in the natural mode of life, or in conditions when synthetic ribosomes take over all the protein production. By manipulating the properties of a particular population of ribosomes, it will be possible to learn much more about the process of protein synthesis.
Literature:
Orelle et al., Protein synthesis by ribosomes with tethered subunits. Nature. 524, 119-124;
Io S.B. and Gy L.O., Synthetic biology: Ribosomal ties that bind. Nature. 524, 45-46.
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18.08.2015