Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli

Abstract

In Escherichia coli, all mRNAs are translated by one pool of functionally identical ribosomes. Here, we describe a system in which a subpopulation of modified ribosomes are directed to a single mutated mRNA species. This was accomplished by changing the Shine-Dalgarno sequence that precedes the heterologous human growth hormone gene from 5' GGAGG to 5' CCTCC or 5' GTGTG. Translation of these modified mRNAs by wild-type ribosomes is very inefficient. When the anti-Shine-Dalgarno region (i.e., the region complementary to the Shine-Dalgarno sequence) at the 3' end of the gene encoding 16S rRNA (rrnB) was altered from 5' CCTCC to 5' GGAGG or 5' CACAC, thus restoring its potential to base-pair with the mutated human growth hormone mRNA, significant expression of this mRNA occurred. Growth hormone synthesis was dependent on induction of the mutated rrnB operon. Subsequently, these specialized ribosomes were made spectinomycin-resistant by the introduction of a C----U substitution at position 1192 of the 16S rRNA. Thus, host protein synthesis could be shut off by the addition of spectinomycin and the specificity and efficiency of the specialized ribosomes could be assessed. Since the specialized ribosomes represent a nonessential subpopulation in the cell, this system offers an approach to the study of mutations elsewhere in the 16S-rRNA gene that otherwise would be lethal to the cell.