Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element.
Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014s(-1). We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.
Mao
Grace
G
Department of Biomedical Engineering, University of California--Irvine, Irvine, CA 92697-2715, USA.
Brody
James P
JP
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D050436:Regulatory Elements, Transcriptional / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D020349:Surface Plasmon Resonance / Q000379:methods*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins