The need for the temporal control of gene expression during development, differentiation or metabolic regulation is fundamental to biology. A primary mechanism for controlling expression of individual genes occurs at the level of initiation of transcription and employs regulatory proteins (transcriptional activators or repressors) that bind to specific DNA control sequences (or operators). These operators are located near the sites where RNA polymerase binds and initiates transcription (promoters); the DNA-protein complexes modulate the efficiency of formation of transcription initiation complexes and thereby control the level of gene expression. Multiple transcriptional regulators are typically involved in regulating the transcription of a particular gene. The level of expression depends on external signals or effectors, and the arrangement of the operators and their associated regulatory proteins on the DNA. Some of the regulatory proteins can act at many different promoters whereas others are promoter specific. These combinatorial mechanisms generate great diversity and can provide over time, coordinate control of many genes as well as fine-tuning of the expression of individual genes. This project addresses the combinatorial control mechanism in a gene family known as the CytR regulon in the bacterium Escherichia coli. Two regulatory proteins working in concert produce both coordinate and differential regulation patterns in seven unlinked genes. The regulatory proteins involved are the global gene regulator CRP and a CytR-specific protein. The interplay between these two proteins and the different arrangements of the respective operator elements at the seven gene promoters of the Cyt regulon underlies this complex combinatorial regulation. This project will employ a multi-disciplinary approach (i) to determine the structures and dynamics of the regulatory proteins and their complexes with DNA, (ii) to probe the effects of interactions between the proteins when bound to DNA and (iii) to develop quantitative understanding of the kinetic mechanisms of transcriptional activation mediated by the specific promoter sequence and the specific protein-protein interactions at that promoter. This will generate a comprehensive view of the molecular mechanisms of combinatorial gene regulation at the structure/function level. Developing this molecular understanding of gene regulation in this bacterial system will have broad impacts on our understanding of not only bacterial physiology, but also on the genetic control of differentiation and development in eukaryotic organisms. The project will also provide multi-disciplinary training for postdoctoral, predoctoral and undergraduate level scientists in structural biology, molecular biophysics, and molecular genetics.
