Investigating the Pyrr-Pyr mRNA Interaction Through Site-Directed Mutagenesis of the PyrR Protein

120 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2001.The pyrimidine biosynthetic (pyr) operon in Bacillus subtilis is regulated by a feedback inhibition loop involving the product of the first gene of the operon, PyrR, and the downstream products of the pathway, uridine nucleotides. Regulation occurs through a transcriptional attenuation mechanism in which PyrR binds in the presence of UMP or UTP to an anti anti-terminator RNA stem-loop and promote early termination of transcription. The goal of this research was to better understand the interaction of the PyrR protein with pyr mRNA and to propose a model for how PyrR binds pyr mRNA. Through site-directed mutagenesis studies six PyrR amino acid residues were identified as being directly involved in RNA binding and one additional amino acid residue was identified as possibly being involved in RNA binding. As a result of in vivo analysis of effects of PyrR mutants on pyrB regulation and electrophoretic mobility shift assays of RNA binding with purified mutant PyrR proteins, threonine 18, histidine 22, arginine 27, arginine 141, arginine 146, and lysine 152 were identified as being directly involved in RNA binding. The involvement of arginine 27 and lysine 152 in RNA binding is not as clearly defined as with the other four amino acids, because mutations at arginine 27 and lysine 152 caused the PyrR protein to be more prone to aggregate. Additionally, arginine 19 was implicated in RNA binding by the effects a mutation in this residue had on regulation of pyrB in vivo. Sequence alignment of pyrR genes from various bacteria identified a strongly conserved region between amino acids 138--156 and a less conserved region between amino acids 15--26. The results of site-directed mutagenesis indicated that the conserved region of PyrR from amino acids 138--156 is an important RNA binding sequence and that additional interactions occur through the more weakly conserved region of PyrR from amino acids 15--26.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD