%0 Generic
%A Gerber, Janina Lara
%C Heidelberg
%D 2026
%F heidok:36944
%K RTCB, RTCB complex, DDX1, CGI-99, FAM98B, ASW, Ashwin, Archease, tRNA ligase
%R 10.11588/heidok.00036944
%T Structural and functional analysis of the  human tRNA ligase complex RTCB
%U https://archiv.ub.uni-heidelberg.de/volltextserver/36944/
%X RTCB-type ligases are a relatively new discovered class of RNA ligases that ligate RNA substrates with 3′-P or 3′-cP and 5′-OH in an GTP and cation dependent manner. In metazoan, RTCB is an essential enzyme that ligates tRNA exons during splicing and is a key component of the IRE1 branch of the unfolded protein response. During the stress response, RTCB ligates the exon halves of the transcription factor XBP1’ mRNA. In humans, RTCB is the catalytic core of the pentameric tRNA ligase complex, consisting of the subunits DDX1, CGI-99, FAM98B and ASW. The function of each component and complex composition are unknown. In archaea and metazoan, RTCB-mediated ligation is dependent on the promoting factor Archease. The mechanism by which Archease enhances RTCB-mediated ligation remained unknown. HsArchease was found to interact with CCAR2, a protein acting in the mTOR pathway. The function and interaction between these two are thus far unknown.  My research focuses on the human tRNA ligase RTCB, the tRNA ligase complex subunits and the promoting factor HsArchease. Here, I use biochemical assays to determine metal ion and nucleotide cofactor dependence on RNA ligation catalysis by HsRTCB in the context of HsArchease’s promoting effect. For the first step of ligation, HsRTCB guanylylation, the human ligase is fully dependent on HsArchease and the cofactors Mn2+ and GTP. Crystal structures of nucleotide-free HsRTCB, monomeric HsArchease and the pre- and post-activation complexes between HsRTCB and HsArchease provide mechanistic details of nucleotide exchange and HsArchease-mediated RTCB activation. In absence of the nucleotide cofactor, HsRTCB adopts an open conformation to enable nucleotide binding in the active site pocket. By formation of a composite three metal ion active site between HsRTCB and HsArchease, electron density shifts facilitate the nucleophilic attack from the HsRTCB active site histidine onto the α-phosphorus atom of GTP. While previous structures of Archease depict a dimeric conformation, the monomeric HsArchease structure as well as the two crystal structures of the pre- and post-activation complex provide evidence that the RTCB ligation promoting form of HsArchease is monomeric, with a conserved tip that fits into the active site pocket of HsRTCB. Furthermore, I purified the human tRNA ligase complex, single subunits and CCAR2 for structural analysis using Cryo-EM or x-ray crystallography. RNA ligation assays demonstrate the acceleration of HsRTCB-mediated ligation by DDX1. In summary, I present crystal structures representing different stages of the HsRTCB ligation cycle and provide mechanistic insights into the HsArchease-dependent activation reaction. Additionally, I structurally analyse the human tRNA ligase complex and the interplay between CCAR2 and HsArchease.