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 i...
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Veröffentlicht in: | Journal of molecular biology 2008-05, Vol.379 (2), p.212-230 |
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Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0022-2836 1089-8638 |
DOI: | 10.1016/j.jmb.2008.03.069 |