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Originally published In Press as doi:10.1074/jbc.M300173200 on March 5, 2003

J. Biol. Chem., Vol. 278, Issue 20, 18628-18637, May 16, 2003
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Signal Recognition Particle Binds to Ribosome-bound Signal Sequences with Fluorescence-detected Subnanomolar Affinity That Does Not Diminish as the Nascent Chain Lengthens*

John J. FlanaganDagger §, Jui-Chang Chen§||, Yiwei Miao, Yuanlong Shao, Jialing Lin**, Paul E. BockDagger Dagger , and Arthur E. JohnsonDagger §§¶¶

From the  Department of Medical Biochemistry and Genetics, Health Science Center, and the Departments of Dagger  Biochemistry and Biophysics and §§ Chemistry, Texas A&M University, College Station, Texas 77843, the ** Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, and the Dagger Dagger  Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

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, Kd 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 (Kd = 71 nM) and RNCs that lack an exposed signal sequence (Kd = 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.

* This work was supported by National Institutes of Health Grants GM 26494 (to A. E. J.) and HL 38779 (to P. E. B.), by Training Grant T32 GM08523 (to J. J. F.), and by the Robert A. Welch Foundation (to A. E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| Present address: Division of Molecular Medicine, Dept. of Medical Research, China Medical College Hospital, Taichung, Taiwan.

¶¶ To whom correspondence should be addressed: College of Medicine, 116 Reynolds Medical Bldg., TAMUS HSC, 1114 TAMU, College Station, TX 77843-1114. Tel.: 979-862-3188; Fax: 979-862-3339; E-mail: aejohnson@tamu.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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