title: Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.
abstract: While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Nikita E
authors.initials: NE
authors.lastname: Chavarria
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Sungmin
authors.initials: S
authors.lastname: Hwang
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Cao
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Xian
authors.initials: X
authors.lastname: Fu
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: Holman
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Dina
authors.initials: D
authors.lastname: Elbanna
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Rodriguez
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Deanna
authors.initials: D
authors.lastname: Arrington
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Markus
authors.initials: M
authors.lastname: Englert
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Sivakumar
authors.initials: S
authors.lastname: Uthandi
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America; Department of Chemistry, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America; Genetics Institute, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Maupin-Furlow
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D019642:RNA, Archaeal / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism; D014529:Uridine / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal; D014452:Ubiquitins; D012343:RNA, Transfer; D046988:Proteasome Endopeptidase Complex; D014529:Uridine