title: Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications.
abstract: Recently, a role for the anticodon wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) has been revealed in the suppression of translational +1 frameshifts in Saccharomyces cerevisiae. Loss of either the mcm5U or s2U parts of the modification elevated +1 frameshift rates and results obtained with reporters involving a tRNALysUUUdependent frameshift site suggested these effects are caused by reduced ribosomal A-site binding of the hypomodified tRNA. Combined loss of mcm5U and s2U leads to increased ribosome pausing at tRNALysUUUdependent codons and synergistic growth defects but effects on +1 frameshift rates remained undefined to this end. We show in here that simultaneous removal of mcm5U and s2U results in synergistically increased +1 frameshift rates that are suppressible by extra copies of tRNALysUUU. Thus, two distinct chemical modifications of the same wobble base independently contribute to reading frame maintenance, loss of which may cause or contribute to observed growth defects. Since the thiolation pathway is sensitive to moderately elevated temperatures in yeast, we observe a heat-induced increase of +1 frameshift rates in wild type cells that depends on the sulfur transfer protein Urm1. Furthermore, we find that temperature-induced frameshifting is kept in check by the dehydration of N6-threonylcarbamoyladenosine (t6A) to its cyclic derivative (ct6A) at the anticodon adjacent position 37. Since loss of ct6A in elp3 or urm1 mutant cells is detrimental for temperature stress resistance we assume that conversion of t6A to ct6A serves to limit deleterious effects on translational fidelity caused by hypomodified states of wobble uridine bases.
authors.affiliation: a Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel , Kassel , Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Klassen
authors.affiliation: a Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel , Kassel , Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Bruch
authors.affiliation: a Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel , Kassel , Germany.
authors.forename: Raffael
authors.identifier: 0000-0001-9484-5247
authors.initials: R
authors.lastname: Schaffrath
mesh_terms: D000926:Anticodon; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016364:Reading Frames; D012270:Ribosomes / Q000378:metabolism*; D013696:Temperature; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D000926:Anticodon; D012343:RNA, Transfer
keywords: 5-methoxycarbonylmethyl-2-thiouridine; cyclic N6-threonylcarbamoyladenosine; tRNA modification; translation; translational frameshift