Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR
- 1 December 2001
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 29 (23), 4851-4865
- https://doi.org/10.1093/nar/29.23.4851
Abstract
The pyrimidine nucleotide biosynthesis (pyr) operon in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR protein binds in a uridine nucleotide-dependent manner to three attenuation sites at the 5'-end of pyr mRNA. PyrR binds an RNA-binding loop, allowing a terminator hairpin to form and repressing the downstream genes. The binding of PyrR to defined RNA molecules was characterized by a gel mobility shift assay. Titration indicated that PyrR binds RNA in an equimolar ratio. PyrR bound more tightly to the binding loops from the second (BL2 RNA) and third (BL3 RNA) attenuation sites than to the binding loop from the first (BL1 RNA) attenuation site. PyrR bound BL2 RNA 4-5-fold tighter in the presence of saturating UMP or UDP and 150- fold tighter with saturating UTP, suggesting that UTP is the more important co-regulator. The minimal RNA that bound tightly to PyrR was 28 nt long. Thirty-one structural variants of BL2 RNA were tested for PyrR binding affinity. Two highly conserved regions of the RNA, the terminal loop and top of the upper stem and a purine-rich internal bulge and the base pairs below it, were crucial for tight binding. Conserved elements of RNA secondary structure were also required for tight binding. PyrR protected conserved areas of the binding loop in hydroxyl radical footprinting experiments. PyrR likely recognizes conserved RNA sequences, but only if they are properly positioned in the correct secondary structure.Keywords
This publication has 37 references indexed in Scilit:
- The Pyrimidine Operon pyrRPB-carA from Lactococcus lactisJournal of Bacteriology, 2001
- Crystal Structure of Toxoplasma gondii Hypoxanthine-Guanine Phosphoribosyltransferase with XMP, Pyrophosphate, and Two Mg2+ Ions Bound: Insights into the Catalytic Mechanism,Biochemistry, 1999
- Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structureJournal of Molecular Biology, 1999
- A 1.4 Å Crystal Structure for the Hypoxanthine Phosphoribosyltransferase of Trypanosoma cruzi,Biochemistry, 1998
- NMR Structure of the Bacteriophage λ N Peptide/boxB RNA Complex: Recognition of a GNRA Fold by an Arginine-Rich MotifCell, 1998
- Interaction of theBacillus stearothermophilusRibosomal Protein S15 with 16 S rRNA: I. Defining the Minimal RNA SiteJournal of Molecular Biology, 1996
- Crystal Structure of the Hypoxanthine−Guanine−Xanthine Phosphoribosyltransferase from the Protozoan Parasite Tritrichomonas foetus,Biochemistry, 1996
- Regulation of levels of purine biosynthetic enzymes in Bacillus subtilis: effects of changing purine nucleotide poolsJournal of General Microbiology, 1991
- Iron(II) EDTA Used to Measure the Helical Twist Along Any DNA MoleculeScience, 1985
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976