ABSTRACT
Transcription is a highly regulated biological process, initiated through the assembly of complexes at the promoter that contain both the general transcriptional machinery and promoter-specific factors. Despite the abundance of studies focusing on transcription, certain questions have remained unanswered. It is not clear how the transcriptional profile of a promoter is affected by genomic context. Also, there is no single cell method to directly compare transcriptional profiles independent of gene length and sequence. In this work, we employ a single genetic site for isolating the transcriptional kinetics of yeast promoters. Utilizing single molecule FISH, we directly compare the transcriptional activity of different promoters, considering both synthesis and cell-to-cell variability. With this approach, we provide evidence suggesting promoters autonomously encode their associated transcriptional profiles, independent of genomic locus, gene length and gene sequence.
Subject(s)
Saccharomyces cerevisiae/genetics , Galactokinase/genetics , Genetic Loci , Heat-Shock Proteins/genetics , In Situ Hybridization, Fluorescence , Microscopy, Fluorescence , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins/genetics , Transcription, Genetic , TranscriptomeABSTRACT
Live-cell imaging of mRNA yields important insights into gene expression, but it has generally been limited to the labeling of one RNA species and has never been used to count single mRNAs over time in yeast. We demonstrate a two-color imaging system with single-molecule resolution using MS2 and PP7 RNA labeling. We use this methodology to measure intrinsic noise in mRNA levels and RNA polymerase II kinetics at a single gene.
Subject(s)
In Situ Hybridization, Fluorescence/methods , RNA, Fungal/metabolism , RNA, Messenger/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/genetics , Gene Expression , Levivirus/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Messenger RNAs undergo 5' capping, splicing, 3'-end processing, and export before translation in the cytoplasm. It has become clear that these mRNA processing events are tightly coupled and have a profound effect on the fate of the resulting transcript. This processing is represented by modifications of the pre-mRNA and loading of various protein factors. The sum of protein factors that stay with the mRNA as a result of processing is modified over the life of the transcript, conferring significant regulation to its expression.
