Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 165 rRNA that are important for the in vitro assembly of the Escherichia coli 305 small ribosomal subunit and for its association with the 505 large ribosomal subunit. The 305 small subunit was reconstituted from phosphorothioate-substituted 165 rRNA and small subunit proteins. Active 305 subunits were selected by their ability to bind to the 505 large subunit and form 705 ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 165 rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 305 subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 305 subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 305 subunit, suggesting that they play a role in the early steps of the 305 subunit assembly.