The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in amino-acyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.EF-Tu.GTP ternary complex formation have been examined spectroscopically. Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val). Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-8. Upon association with EF-Tu.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.GTP. Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu-GTP. Thus, the binding of EF-Tu.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species. It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome. Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNA(Phe), the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group. The Kd of the tRNA(Phe).EF-Tu.GTP ternary complex was determined, at equilibrium, to be 2.6 microM by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein. Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6 degrees C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport.

Protein-RNA and protein-protein interactions involved in the assembly of the signal recognition particle (SRP) were examined using fluorescence spectroscopy. Fluorescein was covalently attached to the 3'-terminal ribose of SRP RNA following periodate oxidation, and the resulting SRP RNA-Fl was reconstituted into a fluorescent SRP species that was functional in promoting translocation of secretory proteins across the membrane of the endoplasmic reticulum. Each of the two protein heterodimers purified from SRP elicited a substantial change in fluorescein emission upon association with the modified RNA. The binding of SRP9/14 to singly-labeled SRP RNA-Fl increased fluorescein emission intensity by 41% at pH 7.5 and decreased its anisotropy from 0.18 to 0.16. The binding of SRP68/72 increased the fluorescein anisotropy from 0.18 to 0.23 but did not alter the emission intensity of SRP RNA-Fl. These fluorescence changes did not result from a direct interaction between the dye and protein because the fluorescein remained accessible to both iodide ions and fluorescein-specific antibodies in the complexes. The spectral changes were elicited by specific SRP RNA-protein interactions, since (i) the SRP9/14- and SRP68/72-dependent changes were unique, (ii) an excess of unlabeled SRP RNA, but not of tRNA, blocked the fluorescence changes, and (iii) no emission changes were observed when SRP RNA-Fl was titrated with other RNA-binding proteins. Each heterodimer bound tightly to the RNA, since the Kd values determined spectroscopically and at equilibrium for the SRP9/14 and the SRP68/72 complexes with SRP RNA-Fl were less than 0.1 and 7 +/- 3 nM, respectively. The binding affinity of SRP68/72 for SRP RNA-Fl was unaffected by the presence of SRP9/14, and hence the binding of the heterodimers to SRP RNA is noncooperative in the absence of SRP54 and SRP19. The SRP protein heterodimers therefore associate randomly and independently with SRP RNA to form domains in the particle that are distinct both structurally and functionally. Any cooperativity in SRP assembly would have to be mediated by SRP54 and/or SRP19.

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