Photolabile oligoDNA probes of Escherichia coli ribosome internal structure

The ribosome is the ribonucleoprotein particle responsible for the biosynthesis of proteins in all cells. This complex of at least 54 proteins and 3 RNA strands, with a combined molecular mass in Escherichia coli of over 2.5 million Daltons, continues to challenge investigators who must use many experimental tools in attempts to elucidate structural and functional features of the ribosome at the molecular level. We have used radioactive, photolabile derivatives of oligoDNAs complementary to E. coli ribosomal RNA (rRNA) in order to examine the spatial arrangement of ribosomal components in the vicinity of rRNA sequences of particular significance for ribosome structure and function. The oligoDNA probes bind to their target sequences in intact ribosomal subunits, and on photolysis incorporate into neighboring ribosomal proteins and rRNA. 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 reverse transcriptase 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 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 the consistencies and inconsistencies with previous models of ribosome structure