Grant: Project Grant

Gene expression is a tightly controlled process that is fundamental to the functioning of cells and organisms. Central to gene expression is transcription in which the enzyme RNA polymerase II (RNAPII) binds to DNA and reads the genetic information from one end of a gene to the other. However, RNAPII does not work alone. Numerous proteins, including transcription factors, interact with and dissociate from DNA in a highly dynamic manner from start to finish, ensuring that genes are expressed appropriately and efficiently. Defects in this process are linked to multiple human diseases, making it essential to understand the nature of protein interactions associated with transcription and how they are coordinated. Studying transcriptional dynamics has been challenging, however, because most existing methods only show a static picture, not how the transcriptional machinery changes over time. It is important, therefore, to develop techniques that can capture these dynamics. We have developed a method called biotin pulse labeling (BPL) to monitor protein interactions in living yeast cells. BPL uses biotin to mark specific protein interactions that occur during any moment. By detecting biotin-labeled proteins, we can examine when specific protein interactions occur and how they change over time. Extending this, we developed BPL-seq, which lets us observe where on the genome these interactions occur. We will apply these methods for examining gene activity and for monitoring the speed at which RNAPII crosses genes, a parameter that recently has come to light as critical regulator of gene expression. Our system will also be used to understand how DNA-bound transcription factors influence the expression of nearby genes, and we will adapt BPL for use in human cells. Overall, our work introduces a powerful new tool for examining how proteins interact over time, offering valuable insights that can improve our understanding of transcription and related diseases.