Published online 25 July 2004
Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved
Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Tsukuba, Ibaraki 305-8566, Japan and 2 Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
* To whom correspondence should be addressed. Tel: +81 29 861 6085; Fax: +81 29 861 6095; Email: pkr-kumar{at}aist.go.jp
Received June 8, 2004; Revised and Accepted July 9, 2004
HutP is an RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus species by binding to cis-acting regulatory sequences on hut mRNA. We recently solved the HutP crystal structure, which revealed a novel fold where three dimers are arranged in a 3-fold axis to form the hexamer. We also identified a minimal RNA binding element sufficient for HutP binding: three UAG trinucleotide motifs, each separated by 4 nt, located just upstream of the terminator. In the present study we have identified important RNA chemical groups essential for HutP interactions, by combining an in vitro selection strategy and analyses by site-specific base substitutions. These analyses suggest that each HutP molecule recognizes one UAG motif, where the first base (U) can be substituted with other bases, while the second and third bases (A and G) are required for the interactions. Further analyses of the chemical groups of the A and G bases in the UAG motif by modified base analogs suggested the importance of the exocyclic NH2 group in these bases. Also, in this motif, only the 2'-OH group of A is important for HutP recognition. Considering the important chemical groups identified here, as well as the electrostatic potential analysis of HutP, we propose that Glu137 is one of the important residues for the HutP–RNA interactions.
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