RESUMEN
Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure and function of its regulatory factor Kti12 have remained elusive. Here, we present the crystal structure of Kti12's nucleotide hydrolase domain trapped in a transition state of ATP hydrolysis. The structure reveals striking similarities to an O-phosphoseryl-tRNA kinase involved in the selenocysteine pathway. Both proteins employ similar mechanisms of tRNA binding and show tRNASec-dependent ATPase activity. In addition, we demonstrate that Kti12 binds directly to Elongator and that ATP hydrolysis is crucial for Elongator to maintain proper tRNA anticodon modification levels in vivo. In summary, our data reveal a hitherto uncharacterized link between two translational control pathways that regulate selenocysteine incorporation and affect ribosomal tRNA selection via specific tRNA modifications.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Trifosfatasas/genética , Procesamiento Postranscripcional del ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Adaptadoras Transductoras de Señales/química , Adenosina Trifosfatasas/química , Anticodón/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Chaetomium/química , Chaetomium/enzimología , Cristalografía por Rayos X , Conformación Proteica , ARN de Transferencia/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Uridina/genéticaRESUMEN
The alpha-tubulin suppressor 1 (ATS1) gene and the killer toxin-insensitive 13 (KTI13) locus from Saccharomyces cerevisiae are allelic. The Ats1/Kti13 gene product interacts with the cell polarity factor Nap1 and promotes growth inhibition of S. cerevisiae by zymocin, a tRNAse toxin complex from Kluyveromyces lactis. Kti13 removal causes zymocin resistance, a trait that is typical of defects in the Elongator complex. Here, we show that Kti13 co-purifies with the Elongator partner protein Kti11 and that the Kti11 interaction, not the Nap1 partnership, requires the C-terminus of Kti13. Moreover, Kti13 functionally relates to roles of the Elongator complex in tRNA wobble uridine modification, tRNA suppression of nonsense (SUP4) and missense (SOE1) mutations and tRNA restriction by zymocin. Also, inactivation of Kti13 or Elongator rescues the thermosensitive growth defect of secretory mutants (sec2-59(ts), sec12-4(ts)), suggesting that Kti13 and Elongator affect secretion processes that depend on the GTP exchange factors Sec2 and Sec12 respectively. Distinct from tandem deletions in KTI13 and Elongator genes, a kti13Delta kti11Delta double deletion induces synthetic sickness or lethality. In sum, our data suggest that Kti13 and Kti11 support Elongator functions and that they both share Elongator-independent role(s) that are important for cell viability.
Asunto(s)
Factores de Elongación de Péptidos/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Regulación Fúngica de la Expresión Génica , Factores Asesinos de Levadura , Kluyveromyces/metabolismo , Mutación , Micotoxinas/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 de Ensamblaje de Nucleosomas , Factores de Elongación de Péptidos/genética , Unión Proteica , ARN de Transferencia/metabolismo , Proteínas Represoras/análisis , Proteínas Represoras/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Supresión GenéticaRESUMEN
The Kluyveromyces lactis killer toxin zymocin insensitive 11 (KTI11) gene from Saccharomyces cerevisiae is allelic with the diphthamide synthesis 3 (DPH3) locus. Here, we present evidence that the KTI11 gene product is a versatile partner of proteins and operates in multiple biological processes. Notably, Kti11 immune precipitates contain Elp2 and Elp5, two subunits of the Elongator complex which is involved in transcription, tRNA modification and zymocin toxicity. KTI11 deletion phenocopies Elongator-minus cells and causes antisuppression of nonsense and missense suppressor tRNAs (SUP4, SOE1), zymocin resistance and protection against the tRNase attack of zymocin. In addition and unlike Elongator mutants, kti11 mutants resist diphtheria toxin (DT), protect against ADP-ribosylation of eukaryotic translation elongation factor 2 (eEF2) by DT and induce resistance against sordarin, an eEF2 poisoning antifungal. The latter phenotype applies to all diphthamide mutants (dph1-dph5) tested and Kti11/Dph3 physically interacts with diphthamide synthesis factors Dph1 and Dph2, presumably as part of a trimeric complex. Moreover, we present a separation of function mutation in KTI11, kti11-1, which dissociates zymocin resistance from DT sensitivity. It encodes a C-terminal Kti11 truncation that almost entirely abolishes Elongator interaction without affecting association with Kti13, another Kti11 partner protein.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Farmacorresistencia Fúngica , Histidina/análogos & derivados , Histidina/farmacología , Indenos/farmacología , Factores Asesinos de Levadura , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación , Micotoxinas/farmacología , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Unión Proteica , Transporte de Proteínas , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron-sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator.
Asunto(s)
Proteínas Bacterianas/química , Histona Acetiltransferasas/química , ARN de Transferencia/química , Dominio Catalítico , Chloroflexi/enzimología , Cristalografía por Rayos X , Unión Proteica , Conformación Proteica en Hélice alfa , Multimerización de Proteína , ARN Bacteriano/química , Especificidad por SustratoRESUMEN
The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer toxin insensitive gene KTI11/DPH3 is involved in the diphthamide modification of eukaryotic elongation factor 2 and, together with Kti13, in Elongator-dependent tRNA wobble base modifications, thereby affecting the speed and accuracy of protein biosynthesis through two distinct mechanisms. We have solved the crystal structures of Saccharomyces cerevisiae Kti13 and the Kti11/Kti13 heterodimer at 2.4 and 2.9 Å resolution, respectively, and validated interacting residues through mutational analysis in vitro and in vivo. We show that metal coordination by Kti11 and its heterodimerization with Kti13 are essential for both translational control mechanisms. Our structural and functional analyses identify Kti13 as an additional component of the diphthamide modification pathway and provide insight into the molecular mechanisms that allow the Kti11/Kti13 heterodimer to coregulate two consecutive steps in ribosomal protein synthesis.