title: The ATP-mediated formation of the YgjD-YeaZ-YjeE complex is required for the biosynthesis of tRNA t6A in Escherichia coli.
abstract: The essential and universal N(6)-threonylcarbamoyladenosine (t(6)A) modification at position 37 of ANN-decoding tRNAs plays a pivotal role in translational fidelity through enhancement of the cognate codon recognition and stabilization of the codon-anticodon interaction. In Escherichia coli, the YgjD (TsaD), YeaZ (TsaB), YjeE (TsaE) and YrdC (TsaC) proteins are necessary and sufficient for the in vitro biosynthesis of t(6)A, using tRNA, ATP, L-threonine and bicarbonate as substrates. YrdC synthesizes the short-lived L-threonylcarbamoyladenylate (TCA), and YgjD, YeaZ and YjeE cooperate to transfer the L-threonylcarbamoyl-moiety from TCA onto adenosine at position 37 of substrate tRNA. We determined the crystal structure of the heterodimer YgjD-YeaZ at 2.3 Å, revealing the presence of an unexpected molecule of ADP bound at an atypical site situated at the YgjD-YeaZ interface. We further showed that the ATPase activity of YjeE is strongly activated by the YgjD-YeaZ heterodimer. We established by binding experiments and SAXS data analysis that YgjD-YeaZ and YjeE form a compact ternary complex only in presence of ATP. The formation of the ternary YgjD-YeaZ-YjeE complex is required for the in vitro biosynthesis of t(6)A but not its ATPase activity.
authors.affiliation: Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS, Bâtiment 430, Université de Paris-Sud, 91405 Orsay Cedex, France.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS, Bâtiment 430, Université de Paris-Sud, 91405 Orsay Cedex, France Sorbonne Universités, UPMC Univ Paris 06, UFR 927, Sciences de la vie, F-75005 Paris, France herman.van-tilbeurgh@u-psud.fr.
authors.forename: Bruno
authors.initials: B
authors.lastname: Collinet
authors.affiliation: Institut de Génétique et de Microbiologie, Université Paris-Sud, UMR8621-CNRS, 91405 Orsay, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Perrochia
authors.affiliation: Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS, Bâtiment 430, Université de Paris-Sud, 91405 Orsay Cedex, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Durand
authors.affiliation: Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, CNRS, Bâtiment 430, Université de Paris-Sud, 91405 Orsay Cedex, France herman.van-tilbeurgh@u-psud.fr.
authors.forename: Herman
authors.initials: H
authors.lastname: van Tilbeurgh
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000378:metabolism; D012343:RNA, Transfer / Q000096:biosynthesis*
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C512523:YeaZ protein, E coli; C560833:YgjD protein, E coli; C500230:YjeE protein, E coli; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer