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tRNA synthetases as potential RNA capping enzymes
Říha, Jan ; Macíčková Cahová, Hana (advisor) ; Brázdovič, Filip (referee)
(EN) A novel type of non-canonical 5′ RNA caps, dinucleoside polyphosphate RNA caps, has recently been discovered in our laboratory. To elucidate the physiological role of these caps, more detailed information about their synthesis is essential. It has been already described that dinucleoside polyphosphate RNA caps (NpnN-RNA caps) can be accepted by RNA polymerase during transcription as a non-canonical initiating nucleotide (NCIN). However, the possibility that NpnN-RNA caps are created post- transcriptionally cannot be ruled out. The best candidates for post-transcriptional capping enzymes are aminoacyl-tRNA synthetases, which, besides their crucial role in translation, are responsible for the synthesis of free dinucleoside polyphosphates. In this thesis, four E. coli tRNA synthetases have been selected, cloned into plasmids, and purified using fast protein liquid chromatography (FPLC). Subsequently, selected tRNA synthetases have been tested for the production of free dinucleoside polyphosphate. These experiments have identified the optimal conditions for production of free dinucleoside polyphosphates, diadenosine tetraphosphate (Ap4A) particularly. The tRNA synthetases were then tested for their capabilities to form an RNA cap on in vitro transcribed radioactively labelled RNA. We found that...
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