Gene expression in the absence of Topoisomerases I and II in S. cerevisiae.
Maria L. Garcia Rubio, Andres Aguilera
Genetics, University of Seville, Av. Reina Mercedes 6, Seville, 41012, Spain
Eukaryotic transcription is a tightly controlled process responsible for the production of a pre-mRNA molecule that undergoes 5'-end capping, splicing, 3'-end cleavage and polyadenylation before being transported to the cytoplasm. Transcription and most of the pre-mRNA processing steps are physically and functionally coupled, which is partially achieved by the C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (RNAPII). However, transcription can also be influenced by a number of structural elements, including DNA supercoiling and chromatin structure. Transcription lead to local changes in DNA supercoiling, which demands the action of DNA topoisomerases to re-establish the normal negative supercoiling of the transcribed DNA. To get further insight into the role that DNA topology might have on transcription we have constructed a yeast transcription assay based on two convergent transcription units. One unit is based on the lacZ gene and the other on the LYS2 gene, each one under the control of a different regulated promoter, GAL1 and tet. The systems are located in plasmids and do not contain transcription terminator regions. As controls we have used similar constructs containing one single transcription unit. Extensive Northern analyses in mutants, such as top1, top2, hpr1, etc, under different expression conditions have permitted us to study how different DNA supercoiling states affect transcription, in particular at the elongation level.