INHIBITION OF TRANSLATIONAL INITIATION IN SACCHAROMYCES-CEREVISIAE BYSECONDARY STRUCTURE - THE ROLES OF THE STABILITY AND POSITION OF STEM-LOOPS IN THE MESSENGER-RNA LEADER

A new modular gene-expression system for use in studies of translation al control in Saccharomyces cerevisiae was constructed. A GAL::PGK fus ion promoter (GPF) directed the inducible synthesis of mRNAs initiated at a single major site. A series of leader sequences were tested in c ombination with each of two reporter genes (encoding chloramphenicol a cetyl transferase (cat) and luciferase (luc)). Stem-loop structures of three different sizes and predicted stabilities were inserted- into e ach of two different unique restriction sites in the leader. After cor rection for relative mRNA abundance, a stem-loop of predicted stabilit y equivalent to approximately -18kcal mol-1 inhibited translation by u p to 89%. The degree of inhibition exerted by the other stem-loops cor related positively with their predicted stabilities. Combinations of t wo stem-loops at different sites yielded an inhibitory effect greater than that of either individual stem-loop alone. Similar inhibitory eff ects were observed with both reporter genes. However, inhibition of tr anslation, particularly of the cat gene, was more effective when the s tem-loop was positioned close to the start codon rather than at the 5' end of the leader. The observed results reflect an important form of post-transcriptional control that is expected to act on a large number of genes in yeast.