Signal Recognition Protein 54 Homologue from Pyrococcus horikoshii Can Bind to GTP and E. coli 4.5S RNA Helix 8

　

Feng-wen CUI^a, Hiroyasu ISHIDA^a, Kazuhiko ISHIKAWA^a, Yoshitsugu KOSUGI^a, Yoshinori KOYAMA^a, Eriko MATSUI^a, Satoko KAWASAKI^a, Yutaka KAWARABAYASHi^{a, b}, and Ikuo MATSUI^*a

　

^a National Institute of Bioscience and Human-technology, Higashi 1-1, Tsukuba, Ibaraki 305, Japan

^b National Institute of Technology and Evaluation, MITI, Nishihara, Shibuyaku, Tokyo, Japan

　

Signal recognition particle 54 kDa protein (SRP54) homologue was identified in Pyrococcus horikoshii genome. Sequence alignment shows that Pyrococcus SRP54 (SRP54^ph) has high similarities to other characterized eucaryotic and prokaryotic SRP^S. SRP54ph and a mutant lacking M domain, SRP54NGph, were expressed as hexa-histidine tagged protein and purified. The overexpression of SRP54ph in E. coli had an inhibitory effect on the cell growth after induction. The digestion patterns of the purified SRP54ph and SRP54NGph with V8 protease were compatible to the typical multi-domain structure observed in other characterized SRPs. The melting temperature (Tm) of SRP54ph and SRP54NGph in GTP binding state are 91.8℃ and 94.1℃, respectively, indicating the thermophilic characteristics of these proteins. In gel retardation and filter binding assays, SRP54ph efficiently and specifically binds to helix8 portion of E. coli 4.5S RNA with a 0.043 mM Kd value under low salt condition. The GTP binding activity of SRP54ph was also proved by photoaffinity cross-linking. These results suggest the conceivable function of SRP54ph for the SRP targeting pathway in Pyrococcus cells.

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