Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency

Abstract

The thymidine kinase gene of herpes simplex virus 1 was mutated by inserting oligodeoxynucleotide linkers into the region of the gene corresponding to the 5' untranslated portion of the mRNA. These linkers, when transcribed into mRNA, might be expected to form hairpin loops and hence to increase the secondary structure of the 5' end of the mRNA. Thymidine kinase insertion derivatives were examined in vivo and in vitro to determine translational efficiency. For the in vivo studies, thymidine kinase insertion derivatives were transfected into thymidine kinase deficient L cells alone and together with a selectable dominant marker, or were assayed in the COS-1 transient expression system. For in vitro studies, thymidine kinase insertion derivatives were subcloned into pSP64. Capped transcripts were analyzed for their ability to bind ribosomes and translate in rabbit reticulocyte lysates and wheat-germ extracts. The results demonstrate that translation efficiency is decreased as the number of linkers is increased and support the view that excessive secondary structure at the 5' end of eukaryotic mRNA impedes translation.