Studies on the role of aminoacyl-tRNA synthesis in the regulation of ribosomal and exo-ribosomal protein synthesis in pathogens

• Main Author: Αποστολίδη, Μαρία
• Other Authors: Σταθόπουλος, Κωνσταντίνος
• Format: Thesis
• Language: English
• Published: 2016
• Subjects: Staphylococcus aureus; T-box riboswitch; Glycyl-tRNA synthetase; Transcription regulation; T-box ριβοδιακόπτης; Συνθετάση glycyl-tRNA; Ρύθμιση της μεταγραφής; 572.886

During the flow of the genetic information, tRNA molecules hold a central position as adaptors between nucleic acids and proteins. Although until recently it was believed that tRNAs act only as passive carriers of amino acids, recent discoveries brought them into spotlight as essential regulators of transcription and translation. It has been recently established that the functional role of tRNA molecules extends beyond their core cellular role in translation. This notion triggered a new point of view of tRNAs and established their involvement in gene expression regulation. The parallel identification of an important riboswitch class which is highly distributed among bacterial organisms (mostly in pathogens), termed T-boxes, confirmed the ability of tRNAs to control essential metabolic pathways. T-box riboswitches are found in the 5’UTR of mRNAs and can utilize either charged or uncharged tRNA molecules as ligands in order to control the expression of enzymes involved in amino acid biosynthesis and aminoacyl-tRNA synthesis. It is known that T-boxes can regulate downstream gene transcription by adopting two alternative conformations termed "terminator" and "antiterminator”. The uncharged tRNA which is initially recognized by the specifier loop in stem I region through codon-anticodon complementarity, stabilizes the antiterminator conformation and as a consequence allows the transcription “read-through” of the downstream gene or operon. In staphylococci, a T-box riboswitch precedes the glyS gene encoding glycyl-tRNA synthetase (GlyRS). GlyRS mediates the formation of the Gly-tRNAGly molecules that serve as substrates for the protein synthesis and for the exo-ribosomal glycine-mediated stabilization of the bacterial cell wall. Previous work of our group revealed that in S. aureus there are two encoded proteinogenic tRNAGly isoacceptors [P1(GCC) and P2(UCC)] and three non-proteinogenic tRNAGly isoacceptors [NP1(UCC), NP2(UCC) and NEW(UCC)] with extra-translational roles which bind poorly to EF-Tu. In the present study, we tried to unravel and verify both the glyS T-box structure and the differential utilization of tRNAGly isoacceptors either in protein synthesis or cell wall formation. Extended bioinformatic and biochemical analyses revealed the existence of a functional T-box regulatory element upstream the glyS gene albeit with divergent structural features in comparison with other known glyQS T-boxes. The most intriguing structural feature identified is the additional 42 nt long intervening sequence, termed stem Sa, which is present in both terminator and antiterminator conformations and moreover, seems to be staphylococci-specific. In vitro binding and transcription readthrough experiments revealed that this T-box riboswitch can utilize both proteinogenic and non-proteinogenic tRNAGly isoacceptors through specifier’s codon unconventional reading. Moreover, in vivo readthrough experiments confirmed this ambiguity and verified the proposed species-specific regulatory mechanism. Additional in vivo data suggested that all tRNAGly isoacceptor presence is essential for growth and viability. In conclusion, specific utilization of different tRNAGly isoacceptor during pathogen’s life contributes both in regulation and synchronization of ribosomal and exo-ribosomal peptide synthesis in a species-specific context. However, the exact regulatory mechanisms that occur during pathogens’s metabolic adaptation and infection require further experimentation. Finally, this study gives for the first time evidence to the existence of an elegant mechanism that synchronizes essential metabolic pathways in pathogens and moreover can be used as an alternative to the current therapy target for the development of novel antimicrobial drugs. In conclusion, the present thesis contributes towards the elucidation of the regulatory role of tRNA molecules, expands our current knowledge on the structure and function of regulatory RNAs in bacteria and underlines the impressive complexity of networks and components of translation during regulation of the flow of the genetic information.