RESUMO
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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mutação , RNA Ribossômico 5S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , Proteínas Ribossômicas/química , Ribossomos/químicaRESUMO
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.
Assuntos
Proteínas de Bactérias/química , RNA Bacteriano/química , RNA Ribossômico 5S/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Genes Bacterianos , Dados de Sequência Molecular , Ligação Proteica/genética , Estrutura Terciária de Proteína , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/química , RNA Ribossômico 5S/química , Proteínas de Ligação a RNA/química , Proteínas Ribossômicas/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Evolução Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
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.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , RNA Ribossômico 5S/metabolismo , Sequência de Aminoácidos , Bactérias/citologia , Proteínas de Bactérias/química , Sequência de Bases , Dados de Sequência Molecular , Ligação Proteica , RNA Ribossômico 5S/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.