In Vivo Analysis of Saccharomyces cerevisiae COX2 mRNA 5′-Untranslated Leader Functions in Mitochondrial Translation Initiation and Translational Activation

Abstract

Abstract We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5′-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator, Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5′-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement. The phenotypes of these mutants delimit a 31-nucleotide segment, from −16 to −46, that contains several short sequence elements necessary for COX2 5′-UTL function in translation. The sequences from −16 to −47 were shown to be partially sufficient to promote translation in a foreign context. Analysis of revertants of both the series of linker-scanning alleles and two short deletion/insertion alleles has refined the positions of several possible functional elements of the COX2 5′-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5′-UTLs that is a potential rRNA binding site.