Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens.
The binding of signal recognition particle (SRP) to ribosome-bound signal sequences has been characterized directly and quantitatively using fluorescence spectroscopy. A fluorescent probe was incorporated cotranslationally into the signal sequence of a ribosome.nascent chain complex (RNC), and upon titration with SRP, a large and saturable increase in fluorescence intensity was observed. Spectral analyses of SRP and RNC association as a function of concentration allowed us to measure, at equilibrium, K(d) values of 0.05-0.38 nm for SRP.RNC complexes with different signal sequences. Competitive binding experiments with nonfluorescent RNC species revealed that the nascent chain probe did not alter SRP affinity and that SRP has significant affinity for both nontranslating ribosomes (K(d) = 71 nm) and RNCs that lack an exposed signal sequence (K(d) = 8 nm). SRP can therefore distinguish between translating and nontranslating ribosomes. The very high signal sequence-dependent SRP.RNC affinity did not decrease as the nascent chain lengthened. Thus, the inhibition of SRP-dependent targeting of RNCs to the endoplasmic reticulum membrane observed with long nascent chains does not result from reduced SRP binding to the signal sequence, as widely thought, but rather from a subsequent step, presumably nascent chain interference of SRP.RNC association with the SRP receptor and/or translocon.
Flanagan
John J
JJ
Department of Medical Biochemistry and Genetics, Health Science Center, Texas A&M University, College Station, Texas 77843, USA.
Chen
Jui-Chang
JC
Miao
Yiwei
Y
Shao
Yuanlong
Y
Lin
Jialing
J
Bock
Paul E
PE
Johnson
Arthur E
AE
D000818:Animals; D001667:Binding, Competitive; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008647:Mesocricetus; D008956:Models, Chemical; D002679:Pan troglodytes; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D051381:Rats; D012270:Ribosomes / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D008024:Ligands; D012333:RNA, Messenger; D018271:Signal Recognition Particle; D012343:RNA, Transfer