The use of single-stranded DNA and RNase H to promote quantitative ‘hybrid arrest of translation’ of mRNA/DNA hybrids in reticulocyte lysate cell-free translations

Abstract

Single-stranded cDNA clones complementary to the 5′ end of TMV RNA have been used to explore the conditions necessary for efficient ‘hybrid arrest of translation’ in the reticulocyte lysate. It is shown that incubations of 20 minutes at 60° in 0.1 M KC1 are sufficient to give almost complete arrest of translation using a clone complementary to the 5′-non-coding region and first 171 coding nucleotides of TMV RNA. However, hybrids with DNA complementary to regions of the mRNA downstream of the first AUG gave variable and in some cases almost no arrest of translation in the reticulocyte lysate unless they were first digested with RNase H. A simple and rapid method for giving complete and highly specific arrest of translation of particular mRNAs in complex mixtures has been developed using both cDNA clones and synthetic oligodeoxynucleotides in conjunction with RNase H digestion. Evidence is presented that suggests that ‘hybrid arrest of translation’' in the wheat-germ cell-free system is primarily due to the action of RNase H. When a reticulocyte lysate was doped with 20 U/ml of RNase H, its ability to translate unannealed mRNA was unaffected but it translated DNA/RNA hybrids extremely poorly.