Kinetic Analysis of SPR Binding Data between Raw Nuclear Extract and DNA: Experiments and Simulations.

摘要

Proteins that bind to cis-regulatory elements of DNA in yeast regulate transcription. In order for genes to be expressed, short and specific DNA sites, where proteins bind, called cis-regulatory elements and special protein complexes such as nucleases and transcription factors need to work together properly. A long term goal of genomics is to identify all essential elements of genomes. Combinatorial interactions between protein complexes and DNA function as mechanisms that provide models with principles of their sophisticated biological behavior. Therefore, studying kinetics of DNA sequence recognition offers a potential insight into its molecular mechanism.;The methods needed to develop a high throughput assay to decode the network regulating transcription have been established to work either in our previous work or in the literature. Synchronization of cell cultures by adding alpha factor to cause cells to arrest in G1 phase facilitates the rest of the entire procedure as follows: samples of both nuclear protein and DNA taken, 16 bp dsDNAs synthesized, protein-DNA binding monitored, and mRNA levels measured to link to protein-DNA binding data. Eventually, we are able to understand the gene regulation in transcription process for one particular yeast gene, RNR1.;In this dissertation, we characterized DNA binding by nuclear protein complexes by surface plasmon resonance biosensor, a quantitative biophysical method for kinetic analysis. We constructed different models to delineate nuclear protein-DNA interactions and interpreted the protein binding data to conclude that transcription requires protein bound to a specific site of DNA. On the basis of our analysis, a parallel model for binding affinity and protein conformation on DNA is presented. These findings not only provide a basis for further study of the complex functions of nuclear protein in yeast but also help elucidate gene regulation between nuclear protein and DNA in yeast. Finally, our work established SPR as a sensitive and valuable technique for studying the logic of transcriptional regulation.