PyrR, A BIFUNCTIONAL RNA-BINDING TRANSCRIPTIONAL ATTENUATION PROTEIN AND URACIL PHOSPHORIBOSYLTRANSFERASE Diana R. Tomchick and Janet L. Smith, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907; Robert J. Turner and Robert L. Switzer, Department of Biochemistry, University of Illinois, Urbana, IL 61801
Bacillus subtilis PyrR regulates the transcription of the pyrimidine nucleotide biosynthetic operon (pyr) via an attenuation mechanism, in response to exogenous pyrimidine levels. UMP-bound PyrR effects regulation at three antiterminator/terminator points in the pyr mRNA by binding to the 5' end of an antiterminator stem-loop structure, which permits the formation of the downstream factor-independent transcription terminator stem-loop. Transcription is thus converted from a readthrough mode with expression of the downstream biosynthetic genes, to a termination mode with reduced expression of the downstream genes. An approximately 50-nucleotide conserved sequence has been identified as the PyrR binding site to the pyr mRNA. While PyrR displays no recognized RNA-binding sequence motif, it contains a short sequence that is characteristic of PRPP binding enzymes, and it is a uracil phosphoribosyltransferase. The relevance of this enzymatic activity to transcriptional attenuation is as yet unknown.
Structures of PyrR in two oligomerization states will be presented. A dimeric form of the enzyme was obtained as a Sm3+ co-crystal. The structure was solved via MAD from data collected at energies around the LII edge of Sm on beamline 19 at the ESRF. Initial phasing was via the program MLPHARE and the atomic model was refined to 1.6Å. The physiologically relevant hexameric form of PyrR crystallized in the space group R32 (dmin of 2.3 Å) and was solved via molecular replacement. The structure of a Mg2+/UMP complex of PyrR will also be presented.
An analysis of the potential RNA-binding site(s) of PyrR and mechanism of transcriptional attenuation will be presented, as well as a comparison of the enzyme to other phosphoribosyltransferase structures.
Supported by NIH grants DK-42303 to J.L. Smith and GM-47112 to R.L. Switzer, and NIH Training Grant GM-07283 to R.J. Turner.