Project description:We used nAnT-iCAGE – unbiased single-nucleotide resolution method for genome-wide transcription start site (TSS) capture, to produce libraries from Saccharomyces cerevisiae total RNA. Our goal was to investigate S. cerevisiae core-promoters and assess the rules of transcription initiation in BY4741 strain grown in YPD media.
Project description:The initial step of RNA polymerase II (Pol II) transcription involves a large number of transcription factors and arises at multiple sites within most promoters. TFIIH is an essential, multi-subunit transcription factor that assembles on promoter DNA with Pol II and five other general transcription factors (GTFs) to form a pre-initiation complex (PIC) for basal transcription. During transcription initiation, TFIIH melts promoter DNA through the ATPase activity of its Ssl2 subunit. In the model eukaryote Saccharomyces cerevisiae, after DNA melting, Pol II scans downstream for usable transcription start sites (TSSs). To understand the function of Ssl2/TFIIH in promoter scanning and TSS selection, we identified novel alleles of SSL2 in genetic screens for mutants defective in TSS distribution that may potentially arise from altered scanning. Consistent with this notion, these ssl2 alleles alter scanning in ways that are distinct from how changes to the Pol II active site alter scanning and this difference is observed genome-wide. Our investigations support two major pathways in controlling promoter scanning and TSS selection, one controlling the efficiency of initiation through Pol II activity or factors regulating Pol II activity; another network appears to control the processivity of scanning by Ssl2/TFIIH.
Project description:Extensive transcriptional heterogeneity revealed by isoform profiling Application of TIF-Seq (Transcript IsoForm Sequencing) to S.cerevisiae. The method was applied to simultaneously identify the 5' capped mRNA transcription start site and the 3' polyadenylation site in different conditions: WT cells grown in glucose media [ypd, 2 biological replicates (bio) and 3 independent library preparations, technical replicates(lib)], WT cells grown in galactose media [ypgal, 4 biological replicates (bio) and 3 independent library preparations, technical replicates(lib)]. A modified protocol designed to enrich in long mRNA molecules was performed for WT cells grown in glucose media [ypd, 2 biological replicates (bio)] and in galactose media [ypgal, 2 biological replicates (bio)] conditions. Finally, control samples performed with a modified protocol designed to identify non-capped but polyadenylated molecules was performed in WT cells grown both in glucose (nypd) and galactose (nypgal) media.
