The long-term goal of this project is to understand the molecular mechanisms which accomplish and regulate protein biosynthesis and secretion, and the reasons why those mechanisms occasionally fail. This requires a knowledge of both the molecular architecture of the various multicomponent complexes that mediate those processes and the nature of the conformational changes that control the functional state of the system. The primary goals of the proposed research are: (i) to determine the magnitude of the structural change which is associated with aminoacyl-tRNA (aa-tRNA) recognition by the ribosome and is coupled to the hydrolysis of a GTP molecule by elongation factor Tu (EF-Tu); (ii) to the interaction between the ribosome and the signal recognition particle (SRP), a novel protein-nucleic acid complex which regulates the synthesis of proteins destined for export; and (iii) to examine environment of the nascent chain and identify its interactions during secretion. Singlet-singlet energy transfer between two fluorescent-labeled tRNAs bound to the same ribosome will be measured before and after EF-Tu-dependent GTP hydrolysis to determine the extent of the molecular movement which is associated with a free energy-releasing process during aa-tRNA recognition. These results will be correlated with other energy transfer data, chemical crosslinking data, and immunoelectron microscopy results to locate the aa-tRNA and EF-Tu binding sites on the ribosome. The topology of the SRP ribosome complex will be examined using energy transfer between various pairs of fluorescent-labeled SRP and ribosomal complex components. Any spectral changes which accompany the association of SRP with the ribosome will be used to determine the thermodynamics and kinetics of the interaction. A unique class of functional aa-tRNA analogs will be used to synthesize specific nascent chains with a fluorescent or photoreactive probe at a defined location, and the region occupied by the nascent chain will be systematically examined, particularly its environment as it passes through the lipid bilayer. The distance of the nascent chain probes from the phospholipids in the membrane and from the inner vesicle surface will be determined by singlet-singlet energy transfer.
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