Ffh and FtsY in a Mycoplasma mycoides signal-recognition particle pathway: SRP RNA and M domain of Ffh are not required for stimulation of GTPase activity in vitro.
Mycoplasma mycoides contains a signal-recognition particle (SRP) composed of an RNA molecule and an SRP54 homologue (Ffh). We have now identified a mycoplasma homologue to the alpha subunit of the mammalian SRP receptor and Escherichia coli FtsY. The protein (MmFtsY) was expressed in E. coli and purified to homogeneity. MmFtsY has a weak intrinsic GTPase activity but GTP hydrolysis was markedly stimulated when it was combined with mycoplasma Ffh (MmFfh) and SRP RNA. Also, in the absence of SRP RNA GTPase activity was significantly enhanced. Furthermore, GTP hydrolysis was stimulated when MmFtsY was combined with the N-terminal GTPase domain (N+G) of MmFfh. These findings indicate that basic features of the GTPase activation mechanism are independent of the C-terminal M domain of the MmFfh protein. We propose that the activation is mediated to a large extent by contacts between the GTPase domains of the mycoplasma Ffh and FtsY proteins and that the contribution of the M domain and SRP RNA in the activation mechanism is mainly for modifying the conformation of the MmFfh GTPase domain.
Macao
B
B
Department of Medical Biochemistry, Göteborg University, Sweden.
Luirink
J
J
Samuelsson
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009176:Mycoplasma mycoides / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; C087406:FtsY protein, Bacteria; D012329:RNA, Bacterial; D018160:Receptors, Cytoplasmic and Nuclear; D018271:Signal Recognition Particle; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases