Role for both DNA and RNA in GTP hydrolysis by the Neisseria gonorrhoeae signal recognition particle receptor.
The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that P(pilE) DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.
Frasz
Cody
C
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-1101, USA.
Arvidson
Cindy Grove
CG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D036781:Fimbriae Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000378:metabolism*; D012329:RNA, Bacterial; D012335:RNA, Ribosomal / Q000502:physiology*; D018271:Signal Recognition Particle / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*
C409968:4.5S RNA; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D018271:Signal Recognition Particle; D036781:Fimbriae Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases