Reassembly of active 30S ribosomal subunits with an unmethylated in vitro transcribed 16S rRNA

Abstract

A plasmid has been constructed, which contains the 16S ribosomal RNA gene of Escherichia coli immediately downstream from a phage T7 promoter. In vitro transcription of this gene by RNA polymerase of the T7 phage yielded an unmethylated 16S rRNA that could be used for the assembly of functional 30S subunits. These subunits, when assayed in a poly(U)-directed translation assay, were as active as 30S subunits reconstructed with native 16S rRNA. This system opens the possibility of investigating the role of the methylations of the rRNA and the functional consequences of site-directed mutagenesis in a rRNA gene. An application of this system was provided by generating a 16S rRNA transcript shortened by about 30 nucleotides at the 3′ end. This truncated rRNA could be reassembled into 30S subunits, about 70% as active as 30S subunits reconstructed with the full-length 16S rRNA transcript.