In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 25 ( 2000-12-05), p. 13732-13737
Abstract:
The split-ubiquitin assay detects protein interactions in vivo . To identify proteins interacting with Gal4p and
Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of
specific proteins in vivo . A library of genomic Saccharomyces cerevisiae DNA fragments fused to the
N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were
C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue
in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a
native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule
pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between
either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of
DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae .
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.250400997
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2000
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
Bookmarklink