Specialized ribosomes: highly specific translation in vivo of a single targetted mRNA species

Abstract

In previous studies [Hui and de Boer, Proc. Natl. Acad. Sci. USA 84 (1987) 4762–4766; Hui et al., Methods Enzymol. 153 (1987) 432–452], it was shown that efficient translation of the human growth hormone mRNA (h GH) species having an altered Shine-Dalgarno (SD) sequence, 5′-GUGUG-3′, depends on the presence of specialized (spc) ribosomes containing the modified anti-SD (ASD) sequence, 5′-CACAC-3′, near the 3′ end of their 16S rRNA. In spite of the altered ASD sequence, spc ribosomes were not found to be committed exclusively to the translation of the h GH mRNA; no more than 30% of the total amount of protein synthesized by such ribosomes was hGH. Once we replace the coding sequence of the h GH mRNA with that of chloramphenicol acetyltransferase (CAT), the specificity of spc ribosomes for translation of a single targetted mRNA, relative to the endogeneous mRNAs, is greatly enhanced; an estimated 80% of the total amount of protein synthesized by spc ribosomes is CAT. Using the inducible spc ribosome system containing the cat gene, we show that, upon induction, spc ribosomes accumulate in large excess over the number needed for optimal translation of the targetted cat mRNA. Despite the excess, only few spc ribosomes initiate translation on a limited number of endogenous mRNAs. The excessive accumulation of spc ribosomes, which are predominantly present as free 30S subunits, is neither deleterious to the cells, nor does it lead to a feedback inhibition of the synthesis of wild-type ribosomes