The Escherichia coli uhp locus encodes an inducible active transport system responsible for the entry of various sugar phosphates and related compounds. Expression of the transport system is induced at the level of transcription by external glucose-6-phosphate. This system represents a relatively simple example of transmembrane signalling as carried out by members of the so-called two-component regulatory systems. Useful features of this system are that the external signal and the three system-specific regulatory genes have been identified. This project will investigate three major aspects of the regulation of gene expression and the mechanism of transport for which this system provides an excellent model. 1) The transcription activator protein UhpA and its target DNA sequence differ substantially from the well-studied homologues NtrC and OmpR, and might activate transcription in a novel manner. Its mechanism of UhpA action will be investigated by defining its DNA target sequences and its effect on the formation of the transcription initiation complex; by testing the effect of its phosphorylation on DNA binding and transcription activation; and by analyzing the sequence requirements and structure of its DNA-binding domain. 2) A second goal is to define the signal transduction pathway and the role of the two membrane-associated regulatory proteins, UhpB and UhpC. Current genetic evidence suggests that UhpB serves as protein kinase/phosphatase and the UhpC serves to bind external inducer. Evidence will be sought to show whether binding of inducer leads to increased phosphorylation of UhpA and whether the UhpB and C proteins act as a complex for their signalling function. 3) The third portion of this proposal is to explore the novel mechanism of the transport protein UhpT. This protein carries out an electroneutral exchange process to couple the accumulation of sugar phosphates with the downhill efflux of inorganic phosphate. The transmembrane topology of UhpT appears to be similar to that of many ion gradient-coupled transporters and it will be worthwhile to try to characterize the structural basis for the difference in transport mechanism. Various mutagenesis regimens are planned to investigate the role of individual amino acids in transport function and to evaluate the use of suppressor analysis to obtain information about the relative orientation of transmembrane segments. Thus, the central approach during the requested period of support is to exploit the genetic systems we have developed to address the structure and interactions of the membrane-associated or DNA-binding proteins of the Uhp system.
