title: Essential structural elements in tRNA(Pro) for EF-P-mediated alleviation of translation stalling.
abstract: The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA(Pro) affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNA(Pro) is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNA(Pro) isoacceptors and tRNA(fMet) in Escherichia coli, and the D-arm of tRNA(fMet) is essential for EF-P-induced acceleration of fMet-puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm.
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|JST, PRESTO, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Katoh
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen 37077, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Wohlgemuth
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Nagano
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen 37077, Germany.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|JST, CREST, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suga
mesh_terms: D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D059372:Nucleotide Motifs / Q000235:genetics; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D014176:Protein Biosynthesis; D011691:Puromycin / Q000737:chemistry / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000378:metabolism; D012361:RNA, Transfer, Pro / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D010455:Peptides; D012358:RNA, Transfer, Met; D012361:RNA, Transfer, Pro; C020746:factor EF-P; C024988:tRNA, formylmethionine-; C011083:polyproline; D011691:Puromycin