Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem-loops in the mRNA leader.
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 -18 kcal 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-loops 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.
Oliveira
C C
CC
Department of Gene Expression, GBF-Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
van den Heuvel
J J
JJ
McCarthy
J E
JE
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004790:Enzyme Induction / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase