RESUMO
Several GTPases participate in bacterial protein biosynthesis. Initiation factor 2 controls the formation of the ribosomal initiation complex and places initiator fMet-tRNAfMet in the ribosomal P-site. Elongation factors Tu and G are responsible for codon-specific binding of the aminoacyl-tRNA to the A-site, and peptidyl-tRNA to the P-site, respectively, during the elongation phase of protein biosynthesis. Release factor 3, a GTPase which is not ubiquitous, is involved in termination and release of the nascent polypeptide. Other translation factors, including initiation factors 1 and 3, elongation factor Ts, release factors 1 and 2, and ribosomal release factor do not belong to the family of GTP/GDP binding proteins. The guanosine nucleotide binding domains of the GTPases involved in translation are structurally related to the Galpha subunit of heterotrimeric G proteins and to the proteins of the Ras family. We have identified and sequenced all genes coding for translation factors in the extreme thermophile Thermus thermophilus. The proteins were overproduced in Escherichia coli, purified, biochemically characterised and used for crystallisation and structural analysis. Further biochemical investigations were aimed at gaining insight into the molecular mechanism underlying the regulation of the GTPase activity of the translation factors, and to elucidate the role of their ribosomal binding sites in this process.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Biossíntese de Proteínas , Thermus thermophilus/metabolismo , Sequência de Aminoácidos , GTP Fosfo-Hidrolases/química , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Thermus thermophilus/genéticaRESUMO
Two polypeptides resistant against proteolytic digestion were identified in Thermus thermophilus translation initiation factor 2 (IF2): the central part of the protein (domains II/III), and the C-terminal domain (domain IV). The interaction of intact IF2 and the isolated proteolytic fragments with fMet-tRNAfMet was subsequently characterized. The isolated C-terminal domain was as effective in binding of the 3' end of fMet-tRNAf Met as intact IF2. N-Formylation of Met-tRNAfMet was required for its efficient binding to the C-terminal domain. This suggests that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is responsible for the recognition of fMet-tRNAfMet by IF2 during translation initiation. Moreover, it was demonstrated that fMet-AMP is a minimal ligand of IF2. fMet-AMP inhibits fMet-tRNAfMet binding to IF2 as well as the activity of IF2 in the stimulation of ApUpG-dependent ribosomal binding of fMet-tRNAf Met. Specific interaction of fMet-AMP with IF2 was demonstrated by 1H-NMR spectroscopy. These findings indicate that fMet-AMP and the 3' terminal fMet-adenosine of fMet-tRNAfMet use the same binding site on the C-terminal domain of IF2 and imply that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is primarily responsible for the fMet-tRNAfMet binding and recognition by IF2.
Assuntos
Monofosfato de Adenosina/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , N-Formilmetionina/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Thermus thermophilus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , TemperaturaRESUMO
Initiation of protein biosynthesis in bacteria requires three initiation factors: initiation factor 1, initiation factor 2 and initiation factor 3. The mechanism by which initiation factors form the 70S initiation complex with initiator fMet-tRNA(fMet) interacting with the initiation codon in the ribosomal P site and the second mRNA codon exposed in the A site is not yet understood. Here, we present a model for the function of initiation factors 1 and 2 that is based on the analysis of sequence homologies, biochemical evidence and the present knowledge of the three-dimensional structures of translation factors and ribosomes. The model predicts that initiation factors 1 and 2 interact with the ribosomal A site mimicking the structure of the elongation factor G. We present data that extend the mimicry hypothesis to initiation factors 1 and 2, originally postulated for the aminoacyl-tRNA x elongation factor Tu x GTP ternary complex, elongation factor G and release factors.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 1 em Eucariotos/metabolismo , Mimetismo Molecular , Dados de Sequência Molecular , Fator G para Elongação de Peptídeos , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Fator de Iniciação 1 em Procariotos , Fator de Iniciação 2 em Procariotos , Fator de Iniciação 3 em Procariotos , Biossíntese de Proteínas , Homologia de Sequência de AminoácidosRESUMO
The nucleotide sequence of ribosomal 5S RNA from sorrel (Rumex acetosa) was determined. This sequence shows the highest homology to Chenopodiaceae and Cruciferae ribosomal 5S RNA when compared to other dicotyledones' 5S rRNA.
