Structural analysis of the regulatory protein of pyrimidine biosynthesis, PyrR

Preethi Chander, Purdue University

Abstract

Bacteria have evolved diverse mechanisms to control gene expression at the level of transcription and translation. Transcriptional attenuation of the genes of bacterial operons allows control of the extent of transcriptional readthrough in response to environmental signals. In Bacillus species, the expression of genes of the pyr operon, responsible for pyrimidine biosynthesis, is controlled by the product of the first gene of the operon, PyrR. In response to the availability of the uridylate end-product of the pathway, PyrR binds to the elongating mRNA transcript of the operon, controlling the formation of alternate RNA stem-loop structures that terminate further transcription. By virtue of its sequence and structural fold, PyrR belongs to the phosphoribosyltransferase (PRT) class of proteins. Although the protein displays some PRTase activity at elevated pH, under physiological conditions the regulatory, RNA-binding function of PyrR appears to be its primary role. Understanding the molecular basis for the evolution of the regulatory role of PyrR has been the goal of this project. Crystal structures of Bacillus caldolyticus PyrR provide insights into how this regulatory role might be achieved. For the first time, complexes of PyrR with the nucleotides, uridine monophosphate (UMP) and guanosine monophosphate (GMP), and divalent magnesium were obtained. Although the binding of UMP was expected, the association of GMP was a novel finding. The PyrR-nucleotide complexes support a model for dual regulation of PyrR by pyrimidines and purines. Sedimentation velocity experiments suggest that the stoichiometry of PyrR-RNA complex formation is one RNA molecule to one PyrR dimer. Electrophoretic mobility shift assays demonstrate that UMP and GMP have opposite effects on RNA binding to the PyrR dimer. Initial crystals of the PyrR-RNA complex, grown in presence of magnesium and UMP, have been obtained. Based on the structural and biochemical data reported here, as well as precedents from other PRT and transcriptional antitermination proteins, a structural model for the PyrR-RNA complex, as well as the role of regulatory nucleotides, is proposed. Overall, the results with the B. caldolyticus PyrR have increased our understanding of the unique adaptation of a PRTase enzyme for a novel RNA-binding role.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Biochemistry|Biophysics

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