Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primari...Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primarily involved in N^(6)-threonylcarbamoyl adenosine(t^(6)A)modification of transfer RNAs(tRNAs).Recently,it was reported that KEOPS participates in homologous recombination(HR)repair in yeast.To characterize the KEOPS in archaea(aKEOPS),we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus.We show that aKEOPS also possesses five subunits,Pcc1,Kae1,Bud32,Cgi121,and Pcc1-like(or Gon7-like),just like eukaryotic KEOPS.Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex(Kae1-Pcc1-Pcc1-Kae1),suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit.Strikingly,none of the genes encoding aKEOPS subunits,including Pcc1 and Pcc1-like,can be deleted in the wild type and in a t^(6)A modification complementary strain named TsaKI,implying that the aKEOPS complex is essential for an additional cellular process in this archaeon.Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI.These results suggest that aKEOPS plays an essential role independent of the cellular t^(6)A modification level.In addition,archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹCCA tail binding module.Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7.The study also reveals a possible link between the function in t^(6)A modification and the additional function,presumably HR.展开更多
In archaea, the HEL308 homolog Hel308a(or Hjm) is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is ...In archaea, the HEL308 homolog Hel308a(or Hjm) is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet(UV) and methyl methanesulfonate(MMS) treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.展开更多
基金supported by the National Key Research and Development Program of China(No.2020YFA0906800)the National Natural Science Foundation of China(Nos.31970546 and 31670061 to Y.S.,31900055 to Q.H.,31970119 to J.N.,and 31771380 to Q.S.)the State Key Laboratory of Microbial Technology.
文摘Kinase,putative Endopeptidase,and Other Proteins of Small size(KEOPS)is a multisubunit protein complex conserved in eukaryotes and archaea.It is composed of Pcc1,Kae1,Bud32,Cgi121,and Gon7 in eukaryotes and is primarily involved in N^(6)-threonylcarbamoyl adenosine(t^(6)A)modification of transfer RNAs(tRNAs).Recently,it was reported that KEOPS participates in homologous recombination(HR)repair in yeast.To characterize the KEOPS in archaea(aKEOPS),we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus.We show that aKEOPS also possesses five subunits,Pcc1,Kae1,Bud32,Cgi121,and Pcc1-like(or Gon7-like),just like eukaryotic KEOPS.Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex(Kae1-Pcc1-Pcc1-Kae1),suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit.Strikingly,none of the genes encoding aKEOPS subunits,including Pcc1 and Pcc1-like,can be deleted in the wild type and in a t^(6)A modification complementary strain named TsaKI,implying that the aKEOPS complex is essential for an additional cellular process in this archaeon.Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI.These results suggest that aKEOPS plays an essential role independent of the cellular t^(6)A modification level.In addition,archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3ʹCCA tail binding module.Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7.The study also reveals a possible link between the function in t^(6)A modification and the additional function,presumably HR.
基金supported by the grants from the National Natural Science Foundation of China (Nos. 3093002, 31170072 and 31470184 to Y.S.)
文摘In archaea, the HEL308 homolog Hel308a(or Hjm) is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet(UV) and methyl methanesulfonate(MMS) treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.