| RNA polymerase is a key regulatory machine for maintaining cell growth and organ development.There are three types of RNA polymerases in eukaryotes,mainly RNA polymerase â… (RNAPâ… ),RNA polymerase â…¡(RNAPâ…¡),and RNA polymerase â…¢(RNAPâ…¢).RNAPâ…¡ is involved in the transcription of all mRNAs in cells.It is a large complex consisting of 12 subunits with a molecular weight of about 520 KD.Although the structure and function of RNAPâ…¡ are clear,the assembly process of the complex is unknown.RNAPâ…¡ cannot self-assemble in vitro,it proves that the assembly of intracellular polymerase complex requires assembly factors.For a long time,the assembly process of RNAPâ…¡ was unclear because of the lack of identification and functional studies of assembly factors.In the previous stage,the research team elucidated the molecular mechanism of the GPN family member Gpn2 and the RNAPâ…¡ binding protein Rba50 to assemble the RNAPâ…¡ Rpb3 subcomplex.The GPN protein family includes Gpn1,Gpn2,and Gpn3.All three members are conserved essential genes,and knockout was fatal,proving that the functions of the three are not redundant.Studies have shown that the GPN protein family Gpn1 and Gpn2 have a genetic interaction.Therefore,this study mainly researches the function of Gpn1 in RNAPâ…¡ assembly.In this study,Gpn1 protein targeted degradation mutants and temperature sensitive mutants were constructedand.It employed techniques such as immunoprecipitation and yeast two-hybrid to clarify the relationship between Gpn1,other assembly factors and RNAPâ…¡ subunits.When Gpn1,Gpn2,or Gpn3 were rapidly degraded,cytoplasmic retention of the large RNAPâ…¡ subunit Rpb1 was observed,proving that the GPN protein family directly participates in the RNAPâ…¡ biosynthesis process.By screening gpn1ts temperature-sensitive strain and observing the effect of growth defects on RNAPâ…¡assembly,it found that Rpb1 showed significant cytoplasmic retention in gpn1ts strain at limiting temperature.It also directly proves that Gpn1 protein affects the assembly of RNAPâ…¡ in the cytoplasm,Rpb1 stays in the cytoplasm,and RNAPâ…¡ is blocked from entering the nucleus.Through high-copy genetic screening of gpn1ts mutant,it was proved that GPN3 and RBA50 are genetic repressors of the gpn1ts mutant.At the same time,through yeast two-hybrid technology,it was demonstrated that Gpn1,Gpn2,and Gpn3 interact with the assembly factor Rba50(human RPAP1).And Gpn1 and Rba50 interact with the Rpb2 subunit of RNAPâ…¡.In addition,in order to study the relationship between Gpn1 and Gpn3,it was found that there is a direct interaction between Gpn1 and Gpn3,and Gpn3 is degraded when Gpn1 protein is degraded,and Gpn1 is also degraded when Gpn3 is degraded.Therefore,we speculated that Gpn1 may cooperate with Rba50 and Gpn3 in the assembly of RNAPâ…¡.This study demonstrated that Gpn1 interacts with RNAPâ…¡,that Gpn1 mutation and degradation cause cytoplasmic retention of Rpb1 and directly participate in RNA polymerase â…¡ assembly.The synergy between Gpn1 and Rba50 and Gpn3 provides a theoretical basis for studying the molecular mechanism of Gpn1 assembling RNAPâ…¡,and also provides a new idea for comprehensive analysis of RNA polymerase â…¡ assembly process. |
