Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae.
The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.
Krogan
N J
NJ
Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Greenblatt
J F
JF
D002846:Chromatography, Affinity; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D046911:Macromolecular Substances; D020411:Oligonucleotide Array Sequence Analysis; D010449:Peptide Mapping; D010641:Phenotype; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000502:physiology*; D015854:Up-Regulation
D005656:Fungal Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D012333:RNA, Messenger; D014157:Transcription Factors; D012319:RNA Polymerase II