Fragmentation of the ribosome to investigate RNA-ligand interactions

RNA molecules perform a variety of important and diverse functions and, therefore, an understanding of their structure and interaction with proteins and ligands is essential. Large RNA molecules (for example, the ribosomal RNAs) are complex and hence reports describing their fragmentation into functional subdomains has provided a means for their detailed analysis. We present here an in vivo approach to study RNA-ligand interactions. This is based on the concept that an RNA fragment could mimic a drug-binding site present on the intact molecule. Overexpression of the fragment would sequester the drug thereby permitting the continued functioning of the ribosome and, thus, ensuring cell viability. Accordingly, a fragment of 16S rRNA encompassing the spectinomycin-binding domain in helix 34 (nucleotides 1046-1065 and 1191-1211) was cloned and in vivo expression resulted in drug resistance. Furthermore, an RNA fragment lacking flanking sequences to helix 34 was also selected from among a pool of random rRNA fragments and shown to confer spectinomycin resistance. A similar in vitro approach is also described for the analysis of rRNA molecules that interact with the yeast elongation factor 3 (EF-3).