tRNA synthetases as potential RNA capping enzymes
| Thesis title in Czech: | tRNA syntetázy jako potenciální RNA čepičkovací enzymy |
|---|---|
| Thesis title in English: | tRNA synthetases as potential RNA capping enzymes |
| Key words: | tRNA syntetázy, RNA čepičkování, prokaryotická RNA, RNA modifikace |
| English key words: | tRNA synthetase, RNA capping, prokaryotic RNA, RNA modifications |
| Academic year of topic announcement: | 2022/2023 |
| Thesis type: | diploma thesis |
| Thesis language: | angličtina |
| Department: | Department of Cell Biology (31-151) |
| Supervisor: | Ing. Hana Macíčková Cahová, Ph.D. |
| Author: | hidden - assigned and confirmed by the Study Dept. |
| Date of registration: | 26.10.2022 |
| Date of assignment: | 26.10.2022 |
| Confirmed by Study dept. on: | 06.02.2023 |
| Date of electronic submission: | 29.04.2024 |
| Date of proceeded defence: | 03.06.2024 |
| Opponents: | Mgr. Filip Brázdovič, Ph.D. |
| References |
| Hudeček, O., et al. Dinucleoside polyphosphates act as 5’-RNA caps in bacteria. Nature Communications, 11, 1052, 2020 Benoni R., et al. Dinucleoside Polyphosphates as RNA building Blocks with Pairing Ability in Transcription Initiation. ACS Chemical Biology, 15, 7, 1765–1772, 2020 Rubio Gomez, M. A. & Ibba, M. Aminoacyl-tRNA synthetases. RNA,26, 910, 2020 Fraga, H. & Fontes, R. Enzymatic synthesis of mono and dinucleoside polyphosphates. Biochimica et Biophysica Acta,1810, 1195, 2011 |
| Preliminary scope of work |
| V naší skupině jsme objevili novou třídu RNA čepiček – dinukleosid polyfosfátů (NpnN) v bakteriích. Ačkoliv je RNA polymeráza může inkorporovat jako první nekanonický iniciační nukleotid do RNA při RNA transkripci, nemůžeme vyloučit možnost, že jsou tyto 5‘ struktury formovány pomocí tzv. čepičkovacích enzymů. Prozatím nebyly identifikovány žádné takové enzymy v bakteriích. Nicméně tRNA syntetázy jsou schopné produkovat volné dinukleosid polyfosfáty. Předpokládáme, že by mohly potenciálně formovat dinukleosid polyfosfátové čepičky přímo na RNA. V tomto projektu student prověří právě tuto možnost, že by tRNA syntetázy mohly fungovat jako 5’ RNA čepičkovací enzymy. Student bude subklonovat několik tRNA syntetáz z E. coli cDNA knihovny do bakteriálních expresních vektorů. Tyto vektory pak použije pro produkci rekombinantních bakteriálních proteinů a vyčistí je pomocí His trap na FPLC. Dále bude testovat enzymovou aktivitu těchto proteinů na malých molekulách a na radioaktivně značené RNA. |
| Preliminary scope of work in English |
| In our search for new RNA modifications, we have discovered entirely new class of 5‘ caps – dinucleoside polyphosphates (NpnN) in bacteria. Eventhough, RNA polymerase can incorporate them as first non-canonical initiating nucleotides in RNA during transcription, we cannot exclude possibility that these 5‘ RNA structures are formed by capping enzymes. There have not been identified any RNA capping enzymes in bacteria. Nevertheless, tRNA synthetases are able to produce free dinucleoside polyphosphates. Therefore, we assume that they can potentially form NpnN RNA caps directly on RNA. In this project, student will explore possibility that tRNA synthetase can work as 5‘ RNA capping enzymes. Student will subclone several tRNA synthetases from E. coli cDNA library into bacterial expression vectors. He will use these vectors for production of recombinant bacterial proteins and he will use His trap for purification of these enzymes on FPLC. Further, he will explore enzymatic activity of purified tRNA synthetases on small molecules and on radioactively labeled RNA. |