Purification and crystallization of HutP protein that regulate hut operon of Bacillus cereus

Functional Nucleic Acids Group, AIST* VALWAY Technology Center, NEC Soft Co. Ltd., Tokyo, Japan**
â—‹Balasundaresan Dhakshnamoorthy* Tomoko S. Misono* Gopinath C.B. Subash* Kumar K.R. Penmetcha* Hiroshi Mizuno**

HutP is a 16.2 kDa protein consisting of 148 amino acid residues; HutP also exists in Bacillus species, which includes B. subtilis, B. anthracis, B. cereus and B. halodurans, with 60% sequence identity. Among these species, HutP of B. anthracis and B. cereus are 100% homologous in primary sequence. Sequence comparison of the Bacillus HutP proteins revealed that the C-terminal amino acid residues are more conserved than the N-terminal residues. Interestingly, the HutP protein binds to the terminator region of the hut mRNA and enhances hut structural gene expression only when activated by L-histidine. Several lines of evidences have indicated that HutP regulates the expression of the down-stream genes of the hut operon by an anti-termination mechanism. The terminator regions of hut operon consisted of at least three UAG repeats (1, 2). To test whether these UAG motifs are important for the HutP recognition, within this genus, we have analyzed the RNA-protein interaction studies, using various biochemical approaches among these species, and found that the HutP specifically recognize UAG repeats. In the present studies, we have identified the important chemical groups within the UAG motif of hut mRNA for HutP recognition of Bacillus cereus. Furthermore, we have established protocol for the expression, purification and crystallization of HutP protein of Bacillus cereus. These studies may reveal the detailed understanding of HutP regulation.
1. Kumarevel et al., Nature 434 (2005) 183-191.
2. Kumarevel et al., NAR 33 (2005) 5494-5502.