Role of Protein L25 and Its Contact with Protein L16 in Maintaining the Active State of Escherichia coli Ribosomes in vivo.

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Ribosomal proteins: structure, function, and evolution.

The question concerning reasons for the variety of ribosomal proteins that arose for more than 40 years ago is still open. Ribosomes of modern organisms contain 50-80 individual proteins. Some are characteristic for all domains of life (universal ribosomal proteins), whereas others are specific for bacteria, archaea, or eucaryotes. Extensive information about ribosomal proteins has been obtained since that time. However, the role of the majority of ribosomal proteins in the formation and functioning of the ribosome is still not so clear. Based on recent data of experiments and bioinformatics, this review presents a comprehensive evaluation of structural conservatism of ribosomal proteins from evolutionarily distant organisms. Considering the current knowledge about features of the structural organization of the universal proteins and their intermolecular contacts, a possible role of individual proteins and their structural elements in the formation and functioning of ribosomes is discussed. The structural and functional conservatism of the majority of proteins of this group suggests that they should be present in the ribosome already in the early stages of its evolution.

[The problems of individual respiratory protection in medical workers].

The paper discusses a problem of high occupational and occupation-induced overall morbidity in medical workers and shows that a discrepancy between the existing system for individual protection of the personnel and specific working conditions and hazardous factors is one of the causes. The authors have developed regional guidelines "Enhancing the efficiency of individual respiratory protection in the workers of health care facilities" which provide evidence-based guidelines for the usage of certified, ergonomically acceptable respiratory protective means at the health care facilities. The main task of medical recommendations is to change the established stereotype in using lowly effective gauze bandages and surgical protective facial masks as respiratory protective means at the therapeutic-and-prophylactic institutions for the personnel.

Protein CTC from Aquifex aeolicus possesses a full-sized 5S rRNA-binding domain.

Ribosomal 5S RNA is the only identified target for proteins of the CTC family. All known proteins of this family, except for CTC from Aquifex aeolicus, contain a full-sized 5S rRNA-binding domain. In the present study a mistake in the published A. aeolicus genome is corrected. It has been demonstrated that the ctc gene of this organism encodes the protein with a full-length 5S rRNA-binding domain. This protein binds specifically to the bacterial 5S rRNA. Thereby, our data show that CTC A. aeolicus is not an exception from the other known CTC proteins.

5S rRNA-recognition module of CTC family proteins and its evolution.

The effects of amino acid replacements in the RNA-binding sites of homologous ribosomal proteins TL5 and L25 (members of the CTC family) on ability of these proteins to form stable complexes with ribosomal 5S RNA were studied. It was shown that even three simultaneous replacements of non-conserved amino acid residues by alanine in the RNA-binding site of TL5 did not result in noticeable decrease in stability of the TL5-5S rRNA complex. However, any replacement among five conserved residues in the RNA-binding site of TL5, as well as of L25 resulted in serious destabilization or complete impossibility of complex formation. These five residues form an RNA-recognition module in TL5 and L25. These residues are strictly conserved in proteins of the CTC family. However, there are several cases of natural replacements of these residues in TL5 and L25 homologs in Bacilli and Cyanobacteria, which are accompanied by certain changes in the CTC-binding site of 5S rRNAs of the corresponding organisms. CTC proteins and specific fragments of 5S rRNA of Enterococcus faecalis and Nostoc sp. were isolated, and their ability to form specific complexes was tested. It was found that these proteins formed specific complexes only with 5S rRNA of the same organism. This is an example of coevolution of the structures of two interacting macromolecules.

Bacterial 5S rRNA-binding proteins of the CTC family.

The presence of CTC family proteins is a unique feature of bacterial cells. In the CTC family, there are true ribosomal proteins (found in ribosomes of exponentially growing cells), and at the same time there are also proteins temporarily associated with the ribosome (they are produced by the cells under stress only and incorporate into the ribosome). One feature is common for these proteins - they specifically bind to 5S rRNA. In this review, the history of investigations of the best known representatives of this family is described briefly. Structural organization of the CTC family proteins and their occurrence among known taxonomic bacterial groups are discussed. Structural features of 5S rRNA and CTC protein are described that predetermine their specific interaction. Taking into account the position of a CTC protein and its intermolecular contacts in the ribosome, a possible role of its complex with 5S rRNA in ribosome functioning is discussed.