In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1–A72 whereas the G1–C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G46 and U47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.
Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains that express the mecA gene but are oxacillin susceptible (OS-MRSA; oxacillin MIC =2 mg/L) are increasingly reported. To gain molecular and functional insights on this observation, we focused on additional factors possibly contributing to phenotypic susceptibility. Methods The nucleotide content of mecA, femA, femB and femX genes, which are considered essential for methicillin resistance, was determined in four OS-MRSA clinical isolates and a genetically similar low-level MRSA control (oxacillin MIC 6 mg/L). Gene expression was quantified compared with the low- and a high-level MRSA (MIC 256 mg/L) control. The tertiary structure of Fem proteins was predicted based on protein structure homology modelling, using web-based automated comparative protein modelling. Growth kinetics were tested for the study and control isolates, to determine whether FemXAB mutations lead to reduced fitness. Results Genes mecA, femA, femB and femX were expressed similarly in the study and the control isolates. Mutations in the gene mecA were not present in any isolate. However, several mutations leading to amino acid substitutions in positions possibly affecting Fem enzyme activity were detected in all fem genes. Two OS-MRSA that had no oxacillin heteroresistance had more mutations in the Fem proteins compared with the remaining isolates that were heteroresistant. The low-level MRSA control had considerably fewer mutations. No differences between growth rates of the OS-MRSA and the MRSA controls were observed. Conclusions Accumulation of amino acid changes in Fem proteins might affect intact cell wall synthesis, even though not causing reduced viability, thus contributing to atypical oxacillin responsiveness.
The specific targeting and inactivation of gene expression represents nowadays the goal of the mainstream basic and applied biomedical research. Both researchers and pharmaceutical companies, taking advantage of the vast amount of genomic data, have been focusing on effective endogenous mechanisms of the cell that can be used against abnormal gene expression. In this context, RNA represents a key molecule that serves both as tool and target for deploying molecular strategies based on the suppression of genes of interest. The main RNA-mediated therapeutic methodologies, deriving from studies on catalytic activity of ribozymes, blockage of mRNA translation and the recently identified RNA interference, will be discussed in an effort to understand the utilities of RNA as a central molecule during gene expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.