Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.
Nhp6p is an architectural Saccharomyces cerevisiae non-histone chromosomal protein that bends DNA and plays an important role in transcription and genome stability. We used the split-ubiquitin system to isolate proteins that interact with Nhp6p in vivo, and we confirmed 11 of these protein-protein interactions with glutathione S-transferase pull-down experiments in vitro. Most of the Nhp6p-interacting proteins are involved in transcription and DNA repair. We utilized the ZDS1, PUR5 and UME6 genes, which are repressed by Nhp6p and its interacting partners Rpb4p and Med3p, to study the chromosomal localization of these three proteins in wild-type and gene deletion strains. Nhp6p, Med3p and Rpb4p were found at the promoters of ZDS1, PUR5 and UME6, indicating that the repressing effects the three proteins had on the expression of these three genes had been direct ones. We also found that Med3p inhibited promoter clearance of RNA polymerase II, which contained the dissociable subunit Rpb4p, while Nhp6p recruited Rpb4p to the basal promoters of ZDS1, PUR5 and UME6. Our results further suggest that Rpb4p inhibits transcription initiation but stimulates transcription elongation and that Nhp6p and Med3p regulate gene expression by controlling the local subunit composition of RNA polymerase II.
Xue
Xiaowei
X
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore.
Lehming
Norbert
N
D048868:Adaptor Proteins, Signal Transducing; D002875:Chromosomes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D024001:HMGN Proteins; D006801:Humans; D056892:Mediator Complex; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D024001:HMGN Proteins; D056892:Mediator Complex; C086471:NHP6A protein, S cerevisiae; D009687:Nuclear Proteins; C110281:PGD1 protein, S cerevisiae; D021122:Protein Subunits; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C089979:UME6 protein, S cerevisiae; D025801:Ubiquitin; C101740:ZDS1 protein, S cerevisiae; D012319:RNA Polymerase II; C423309:RPB4 protein, S cerevisiae