Photolabile oligoDNA probes of internal Escherichia coli ribosomal structure.

ABSTRACT

We are examining the spatial arrangement of ribosomal components in the vicinity of rRNA sequences of particular significance for ribosome structure and function through the use of radioactive, photolabile derivatives of oligoDNAs complementary to such sequences. The oligoDNA probes bind to their target sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components. Labeled ribosomal proteins are subsequently identified by SDS-PAGE, RP-HPLC and immunoprecipitation. Labeled rRNA is identified at the nucleotide level by RNase H cleavage and primer extension. The results obtained provide a description of the neighborhood surrounding a particular rRNA sequence and impose important constraints on the evolving three-dimensional models of both the 30S and 50S subunits, since they typically oblige several ribosomal components to fall within defined distances of the targeted rRNA. Our results will be discussed in terms of these models, emphasizing their consistency or inconsistency.