Crystal Structure of the Ribonucleoprotein Core of the Signal Recognition Particle

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A fragment of the E. coli Ffh corresponding to residues 298 to 453 was probed with V8 and trypsin proteases in the presence and absence of RNA under conditions that yield limited cleavage of the protein. Samples of the proteolytic reaction were taken between 5 min and 1 hour and subjected to matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry [

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]. Although the free protein was rapidly proteolyzed (<5 min), binding of RNA protected a region corresponding to residues 318 to 432. Further mapping yielded a minimal M domain comprising residues 328 to 432 that still binds to the 4.5S RNA with the same affinity as that of full-length Ffh. This construct consists of the signal sequence binding region as well as all of the universally conserved amino acids implicated in RNA recognition (Fig. 1D). Cys-406 was mutated to Ser to avoid oxidation that otherwise destroys RNA binding activity (24). In the RNA, the GGAA tetraloop was changed to GAAA to promote RNA-RNA interactions in the crystal lattice.

The apparent dissociation constant (K_d) for this RNA-protein interaction was determined with a modification of the nitrocellulose filterbinding assay. Briefly, trace quantities of 5′ end–labeled RNA (<1 pM) were incubated with varying concentrations of M domain in a buffer containing 20 mM K-Hepes (pH 7.5), 100 mM KCl, 10 mM MgCl₂, 0.5 mM Na-EDTA, tRNA (0.1 mg/ml), and 0.01% Igepal C-630 for 30 min at room temperature. These reaction mixtures were then filtered through a nitrocellulose filter (BA85, Schleicher & Schuell) and a positively charged nylon filter (Hybond+, Amersham). The filters were washed with two 200-μl samples of binding buffer, and the radioactivity corresponding to free and bound quantified with a PhosphorImager (Molecular Dynamics). The fraction of RNA bound was fit to a Langmuir isotherm to yield K_d.

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]. The backbone root mean square deviation (rmsd) between the E. coli and T. aquaticus M domains is 0.68 Å, and 0.66 Å between the E. coli and human M domains.

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Nucleotide analog interference mapping was performed as described [

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]. In brief, phosphorothioate-tagged nucleotide analogs were incorporated randomly into the 4.5S RNA at the 5% level by run-off transcription by T7 RNA polymerase. This RNA was 5′ ³²P end-labeled and incubated with M domain at a concentration of 25 pM under standard binding conditions (23). This reaction was passed through a nitrocellulose filter to select for protein-bound RNA, the retained RNA eluted off the filter, and ethanol precipitated. The RNA pellet was resuspended in 10 μl of doubly distilled H₂O and 10 μl of formamide load buffer, and in parallel with control unselected RNA, the phosphorothioate linkages were cleaved by the addition of 1 μl of 1 mM I₂-ethanol solution and incubation at 90°C for 1 min. The cleavage products were resolved by electrophoresis on a 12% denaturing polyacrylamide gel. The band intensities were quantified by phosphorimaging and the extent of interference determined as described [

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A number of other nucleotide analogs, including 2′ deoxyadenosine, diamino purine, inosine, and N-methyl guanosine, have also been used, and the results are consistent with the crystal structure.

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We thank S. Brown for providing the 4.5S RNA conditionally deficient strains of E. coli and helpful advice; C. Ogata for time and assistance at beamline X4A of the National Synchrotron Light Source, Brookhaven National Laboratory; T. Earnest for support at beamline 5.0.2 of the Advanced Light Source, Lawrence Berkeley National Laboratory; R. Hanna, J. Kieft, A. Luptak, and M. Talavera for help with beamline data collection and useful discussions; P. Adams and L. Rice for assistance with the refinement; the staff of the Yale Center for Structural Biology for crystallographic and computational support; S. Strobel for providing the nucleotide analogs and for helpful discussions; S. Basu, J. Cate, J. Davis, T. Griffin, J. Ippolito, S. Ryder, P. Scamborova, P. Sigler, and T. Steitz for many helpful discussions; and J. Cate, J. Kieft, V. Rath, and S. Strobel for critically reading the manuscript. We are particularly indebted to A. Ferré-D'Amaré for excellent advice and support throughout this project. This project was funded in part by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (R.T.B.), a Howard Hughes Medical Institute (HHMI) summer fellowship (B.R.), and by support from the NIH, HHMI, and the David and Lucile Packard Foundation. J.A.D. is a Fellow of the David and Lucile Packard Foundation and an Assistant Investigator of HHMI. Coordinates of the SRP core and structure factor amplitudes have been deposited in the PDB (accession code 1DUL). Coordinates are also available at www.csb.yale.edu/people/doudna/doudna_people.html.