This project entails the use of photochemical crosslinking techniques to investigate the structure of (a) the peptidyl-and aminoacyl-tRNA binding sites on E. coli ribosomes and (b) the binding sites for ribosomal proteins on E. coli ribosomal RNA. Specifically developed aryl azide reagents are attached non-photochemically to available thio or amino groups in selected tRNA molecules and ribosomal proteins. The modified components are then allowed to form specific complexes with the 70S ribosome or ribosomal RNA, as appropriate. Irradiation results in the photochemical decomposition of the azides to highly reactive nitrenes which can insert into neighboring molecules of the complex. In a number of cases, specific aminoacyl-tRNA molecules have been directly crosslinked to the RNA component of the 30S ribosomal subunit by irradiation with near ultraviolet light. The sites of insertion in ribosomal RNA will be determined by nucleic acid sequencing methods. Crosslinks to proteins will be investigated by chromatography and by one- and two-dimensional gel electrophoresis. The results of these experiments should aid in defining the spatial arrangement of constituents within functional macromolecular aggregates such as ribosomes.