SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting.
The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.
Siu
Fai Y
FY
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.
Spanggord
Richard J
RJ
Doudna
Jennifer A
JA
D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D005816:Genetic Complementation Test; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D021381:Protein Transport; D012329:RNA, Bacterial / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism
C409968:4.5S RNA; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; C087406:FtsY protein, Bacteria; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D018160:Receptors, Cytoplasmic and Nuclear; D018271:Signal Recognition Particle; D020558:GTP Phosphohydrolases