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Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem‐loops in the mRNA leader
Author(s) -
Oliveira Caria C.,
Heuvel Joop J.,
McCarthy John E. G.
Publication year - 1993
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1993.tb01713.x
Subject(s) - biology , translation (biology) , saccharomyces cerevisiae , gene , stem loop , reporter gene , messenger rna , translational regulation , genetics , eukaryotic translation , start codon , microbiology and biotechnology , stem cell , gene expression
Summary A new modular gene‐expression system for use in studies of translational control in Saccharomyces cerevisiae was constructed. A GAL::PGK fusion promoter (GPF) directed the inducible synthesis of mRNAs initiated at a single major site. A series of leader sequences were tested in combination with each of two reporter genes (encoding chloramphenicol acetyl transferase (cat) and luciferase (luc)). Stem‐loop structures of three different sizes and predicted stabilities were inserted into each of two different unique restriction sites in the leader. After correction for relative mRNA abundance, a stem‐loop of predicted stability equivalent to approximately −18kcal mol −1 inhibited translation by up to 89%. The degree of inhibition exerted by the other stem‐loops correlated positively with their predicted stabilities. Combinations of two stem‐l oops at different sites yielded an inhibitory effect greater than that of either individual stem‐loop alone. Similar inhibitory effects were observed with both reporter genes. However, inhibition of translation, particularly of the cat gene, was more effective when the stem‐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.