Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis.
HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP-RNA interactions. The best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+, while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP-RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein-RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.
Kumarevel
Thirumananseri
T
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST) Central 6, Tsukuba, Ibaraki 305-8566, Japan.
Mizuno
Hiroshi
H
Kumar
Penmetcha K R
PK
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001464:Barium / Q000737:chemistry; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D009876:Operon; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D013728:Terminator Regions, Genetic
D001426:Bacterial Proteins; D002413:Cations, Divalent; D008670:Metals; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C064872:antiterminator proteins, Bacteria; D001464:Barium; D008345:Manganese; D006639:Histidine; D008274:Magnesium