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Structural and Functional characterization of flavoenzymes involved in posttranscriptional modification of tRNA

Abstract : Posttranscriptional modification of ribonucleic acids (RNAs) is a crucial maturation step conserved in all domains of life. During my thesis, I have brought structural and functional insights on flavoenzymes involved in transfer RNA (tRNA) modifications: dihydrouridine synthase (Dus) responsible for dihydrouridine formation using flavin mononucleotide (FMN) and TrmFO responsible for C5 methylation of uridine position 54 relying on flavin adenosine dinucleotide (FAD) and methylenetetrahydrofolate. To elucidate the chemical mechanism of TrmFO we designed an apoprotein via a single mutation that could be reconstituted in vitro with FAD. Furthermore, we chemically synthesized the postulated intermediate active species consisting of a flavin iminium harboring a methylene moiety on the isoalloxazine N5 that was further characterized by mass spectrometry and UV-visible spectroscopy. Reconstitution of TrmFO with this molecule restored in vitro activity on a tRNA transcript proving that TrmFO uses FAD as a methylating agent via a reductive methylation.Dus2 reduces U20 and is comprised of a canonical Dus domain however, mammals have an additional double-stranded RNA-binding domain (dsRBD). To bring functional insight for this modular organization, we showed that only full length human Dus2 was active while its isolated domains were not. tRNA recognition is driven by the dsRBD via binding the acceptor and TΨ stem of tRNA with higher affinity then dsRNA as evidenced by NMR. We further solved the X-ray structures for both domains showing redistribution of surface positive charges justifying the involvement of this dsRBD for tRNA recognition in mammalian Dus2. This was attributed to a peculiar N-terminal extension proven by mutational analysis and an X-ray structure of dsRBD in complex with 22-nucleotide dsRNA. Altogether our work illustrates how during evolution, Dus2 enzymes acquired an engineered dsRBD for efficient tRNA binding via a ruler mechanism.
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https://tel.archives-ouvertes.fr/tel-01680795
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Submitted on : Thursday, January 25, 2018 - 1:35:11 PM
Last modification on : Wednesday, September 23, 2020 - 4:34:42 AM
Long-term archiving on: : Friday, May 25, 2018 - 3:56:59 AM

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  • HAL Id : tel-01680795, version 2

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Charles Bou Nader. Structural and Functional characterization of flavoenzymes involved in posttranscriptional modification of tRNA. Biochemistry, Molecular Biology. Université Pierre et Marie Curie - Paris VI, 2017. English. ⟨NNT : 2017PA066205⟩. ⟨tel-01680795v2⟩

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