ABSTRACT
The RNA polymerase II enzyme from the yeast Saccharomyces cerevisiae is a complex of 12 subunits, Rpb1 to Rpb12. Crystal structures of the full complex show that the polymerase consists of two separable components, a 10-subunit core including the catalytic active site and a heterodimer of the Rpb4 and Rpb7 subunits. To characterize the role of the Rpb4/7 heterodimer during transcription in vivo, chromatin immunoprecipitation was used to examine an rpb4Delta strain for effects on the behavior of the core polymerase as well as recruitment of other protein factors involved in transcription. Rpb4/7 cross-links throughout transcribed regions. Loss of Rpb4 results in a reduction of RNA polymerase II levels near 3' ends of multiple mRNA genes as well as a decreased association of 3'-end processing factors. Furthermore, loss of Rpb4 results in altered polyadenylation site usage at the RNA14 gene. Together, these results indicate that Rpb4 contributes to proper cotranscriptional 3'-end processing in vivo.
Subject(s)
Gene Expression Regulation, Fungal , RNA 3' End Processing/physiology , RNA Polymerase II/physiology , Saccharomyces cerevisiae Proteins/physiology , Transcription Factors/physiology , Transcription, Genetic , Chromatin Immunoprecipitation , Dimerization , Protein Interaction Mapping , RNA, Fungal/genetics , RNA, Messenger/genetics , Structure-Activity Relationship , mRNA Cleavage and Polyadenylation Factors/physiologyABSTRACT
The evolutionarily conserved multisubunit THO complex, which is recruited to actively transcribed genes, is required for the efficient expression of FLO11 and other yeast genes that have long internal tandem repeats. FLO11 transcription elongation in Tho- mutants is hindered in the region of the tandem repeats, resulting in a loss of function. Moreover, the repeats become genetically unstable in Tho- mutants. A FLO11 gene without the tandem repeats is transcribed equally well in Tho+ or Tho- strains. The Tho- defect in transcription is suppressed by overexpression of topoisomerase I, suggesting that the THO complex functions to rectify aberrant structures that arise during transcription.
Subject(s)
Genes, Fungal/genetics , Multiprotein Complexes/genetics , Repetitive Sequences, Nucleic Acid , Transcription, Genetic , Cell Adhesion/physiology , Gene Expression Regulation, Fungal , Mutation/genetics , Open Reading Frames/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The budding yeast, Saccharomyces cerevisiae, responds to changes in external osmolarity through the activation of an osmosensing signal transduction pathway. Using lacZ-reporter gene fusions, clonal cell lines were screened for levels of beta-galactosidase activity in the presence or absence of osmotic stress. A screen of 9,000 transformants displayed 663 (7%) gene fusions that were active in rich medium. Each of the transformants were also assayed for gene activity 24 h following a transfer to high osmolarity medium (0.6 M NaCl) and of the 9,000 clonal cell lines, 86 (1%) displayed a decrease in expression, and seven (0.1%) displayed a reproducible increase in gene expression during primary screening. The chromosomal loci of the lacZ insertions were determined, and the gene(s) associated with that site was examined for osmotically induced expression using RNA blot analysis. Five stress-activated genes were analysed by RNA blot: YDL222C, NMD2, PTC7, FAA4 and YRF1. The genes identified by this screen encompass cellular adaptations to stress including signal transduction, protein myristoylation and fatty acid/sphingolipid content in the cell membrane.
