The instability of messenger RNA is crucial to the control of gene expression. In Bacillus subtilis, RNase Y is the major decay-initiating endoribonuclease. Here, we show how this key enzyme regulates its own synthesis by modulating the longevity of its mRNA. Autoregulation is achieved through cleavages in two regions of the rny (RNase Y) transcript: (i) within the first ~100 nucleotides of the open reading frame, immediately inactivating the mRNA for further rounds of translation; (ii) cleavages in the rny 5′ UTR, primarily within the 5′-terminal 50 nucleotides, creating entry sites for the 5′ exonuclease J1 whose progression is blocked around position −15 of the rny mRNA, potentially by initiating ribosomes. This links the functional inactivation of the transcript by RNase J1 to translation efficiency, depending on the ribosome occupancy at the translation initiation site. By these mechanisms, RNase Y can initiate degradation of its own mRNA when the enzyme is not occupied with degradation of other RNAs and thus prevent its overexpression beyond the needs of RNA metabolism.

Two-component systems (TCS) and small regulatory RNAs (sRNAs) are both widespread regulators of gene expression in bacteria. TCS are in most cases transcriptional regulators. A large class of sRNAs act as post-transcriptional regulators of gene expression that modulate the translation and/or stability of target-mRNAs. Many connections have been recently unraveled between these two types of regulators, resulting in mixed regulatory circuits with poorly characterized properties. This study focuses on the negative feedback circuit that exists between the EnvZ-OmpR TCS and the OmrA/B sRNAs. We have shown that OmpR directly activates transcription from the omrA and omrB promoters, allowing production of OmrA/B sRNAs that target multiple mRNAs, including the ompR-envZ mRNA. This control of ompR-envZ by the Omr sRNAs does not affect the amount of phosphorylated OmpR, i.e. the presumably active form of the regulator. Accordingly, expression of robust OmpR targets, such as the ompC or ompF porin genes, is not affected by OmrA/B. However, we find that several OmpR targets, including OmrA/B themselves, are sensitive to changing total OmpR levels. As a result, OmrA/B limit their own synthesis. These findings unravel an additional layer of control in the expression of some OmpR targets and suggest the existence of differential regulation within the OmpR regulon.

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.

5S рРНК-связывающиеся рибосомные белки семейства L25 являются эволюционным приобретением бактерий. Ранее нами было показано, что 1) одиночные замены в РНК-связывающем модуле белка данного семейства приводят к дестабилизации комплекса с 5S рРНК или к невозможности его образования in vitro; 2) AL25 рибосомы Escherichia coli менее эффективны в синтезе белка in vivo, чем контрольные рибосомы. В настоящей работе исследована эффективность встраивания белка L25 E. coli с мутациями в 5S рРНК-связывающем участке в рибосому in vivo. Показано, что мутации в белке L25, лишающие его способности связываться со свободной 5S рРНК, не препятствуют эффективному и правильному встраиванию белка в рибосому. Об этом свидетельствует то, что присутствие даже очень слабо удерживающегося мутантного белка L25 в рибосоме положительно влияет на активность аппарата трансляции in vivo. Это указывает на существование альтернативного пути встраивания данного белка в рибосому, исключающего предварительное образование комплекса с 5S рРНК. В то же время, стабильный контакт с 5S рРНК оказывается очень важен для удержания белка L25 в рибосоме, и в этом ключевую роль играют консервативные остатки РНК-связывающего модуля белка.

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.

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.

In the present work, ribosomes assembled in bacterial cells in the absence of essential ribosomal protein L5 were obtained. After arresting L5 synthesis, Escherichia coli cells divide a limited number of times. During this time, accumulation of defective large ribosomal subunits occurs. These 45S particles lack most of the central protuberance (CP) components (5S rRNA and proteins L5, L16, L18, L25, L27, L31, L33 and L35) and are not able to associate with the small ribosomal subunit. At the same time, 5S rRNA is found in the cytoplasm in complex with ribosomal proteins L18 and L25 at quantities equal to the amount of ribosomes. Thus, it is the first demonstration that protein L5 plays a key role in formation of the CP during assembly of the large ribosomal subunit in the bacterial cell. A possible model for the CP assembly in vivo is discussed in view of the data obtained.

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.

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.

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.

Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 and Thermus thermophilus TL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5·5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.

Chemischer InformationsdienstVolume 7, Issue 6 Physical Inorganic Chemistry ChemInform Abstract: DETERMINATION OF HEATS OF FORMATION OF Γ-IRON(III) OXIDE, α-IRON(III) HYDROXIDE OXIDE, AND Γ-IRON(III) HYDROXIDE OXIDE BY QUANTITATIVE THERMAL ANALYSIS A. V. KOROBEINIKOVA, A. V. KOROBEINIKOVASearch for more papers by this authorV. I. FADEEVA, V. I. FADEEVASearch for more papers by this authorL. A. REZNITSKII, L. A. REZNITSKIISearch for more papers by this author A. V. KOROBEINIKOVA, A. V. KOROBEINIKOVASearch for more papers by this authorV. I. FADEEVA, V. I. FADEEVASearch for more papers by this authorL. A. REZNITSKII, L. A. REZNITSKIISearch for more papers by this author First published: February 10, 1976 https://doi.org/10.1002/chin.197606010AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References A. V. KOROBEINIKOVA, V. I. FADEEVA, L. A. REZNITSKII, DETERMINATION OF HEATS OF FORMATION OF Γ-IRON(III) OXIDE, α-IRON(III) HYDROXIDE OXIDE, AND Γ-IRON(III) HYDROXIDE OXIDE BY QUANTITATIVE THERMAL ANALYSIS, Izv. Akad. Nauk SSSR, Neorg. Mater., 1975, 11, 1856. Google Scholar Volume7, Issue6February 10, 1976 ReferencesRelatedInformation