SPECIALIZED RIBOSOMES - HIGHLY SPECIFIC TRANSLATION IN-VIVO OF A SINGLE TARGETED MESSENGER-RNA SPECIES

In previous studies [Hui and de Beer, Proc, Natl. Acad. Sci. USA 84 (1 987) 4762-4766; Hui et al., Methods Enzymol. 153 (1987) 432-452], it w as shown that efficient translation of the human growth hormone mRNA ( hGH) 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 t heir 16S rRNA. In spite of the altered ASD sequence, spc ribosomes wer e not found to be committed exclusively to the translation of the hGH mRNA; no more than 30% of the total amount of protein synthesized by s uch ribosomes was hGH. Once we replace the coding sequence of the hGH mRNA with that of chloramphenicol acetyltransferase (CAT), the specifi city of spc ribosomes for translation of a single targetted mRNA, rela tive to the endogeneous mRNAs, is greatly enhanced; an estimated 80% o f the total amount of protein synthesized by spc ribosomes is CAT. Usi ng the inducible spc ribosome system containing the cat gene, we show that, upon induction, spc ribosomes accumulate in large excess over th e number needed for optimal translation of the targetted car mRNA. Des pite the excess, only few spc ribosomes initiate translation on a limi ted number of endogenous mRNAs. The excessive accumulation of spc ribo somes, which are predominantly present as free 30S subunits, is neithe r deleterious to the cells, nor does it lead to a feedback inhibition of the synthesis of wild-type ribosomes.