Structure of the nucleotide complex of PyrR, the pyr attenuation protein from Bacillus caldolyticus, suggests dual regulation by pyrimidine and purine nucleotides.
PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60 degrees C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg2+, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg2+ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.
Chander
Preethi
P
Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA.
Halbig
Kari M
KM
Miller
Jamie K
JK
Fields
Christopher J
CJ
Bonner
Heather K S
HK
Grabner
Gail K
GK
Switzer
Robert L
RL
Smith
Janet L
JL
D001407:Bacillus / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D015394:Molecular Structure; D010430:Pentosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011685:Purine Nucleotides / Q000378:metabolism*; D011742:Pyrimidine Nucleotides / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012313:RNA; D010430:Pentosyltransferases; C098851:PyrR protein, bacteria
Nucleic Acids Res. 2001 Dec 1;29(23):4851-65
11726695
Curr Opin Struct Biol. 2001 Dec;11(6):733-9
11751055
J Bacteriol. 2002 May;184(9):2521-8
11948166
J Biol Chem. 2003 Feb 28;278(9):6921-7
12482852
J Bacteriol. 2003 Jul;185(14):4099-109
12837784
Methods Enzymol. 2003;371:392-404
14712717
Proteins. 1991;11(4):281-96
1758883
Methods Enzymol. 1997;277:505-24
18488322
J Bacteriol. 1979 Jan;137(1):82-91
216664
Methods Enzymol. 1997;276:307-26
27754618
J Mol Biol. 1986 May 5;189(1):113-30
3537305
Microbiology. 1994 Mar;140 ( Pt 3):479-91
7516791
J Bacteriol. 1995 Jan;177(1):271-4
7798145
Science. 1994 Jun 3;264(5164):1427-33
8197456
J Bacteriol. 1994 Jun;176(12):3698-707
8206848
Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5428-32
8516284
J Bacteriol. 1996 Dec;178(24):7206-11
8955403
J Bacteriol. 1997 Jun;179(11):3470-81
9171389
Protein Expr Purif. 1997 Aug;10(3):356-64
9268683
Biochemistry. 1997 Sep 2;36(35):10718-26
9271502
Biochemistry. 1997 Sep 16;36(37):11061-8
9333323
J Biol Chem. 1998 Mar 6;273(10):5932-8
9488732
Structure. 1998 Mar 15;6(3):337-50
9551555
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Prog Nucleic Acid Res Mol Biol. 1999;62:329-67
9932459