IN-VIVO ANALYSIS OF SACCHAROMYCES-CEREVISIAE COX2 MESSENGER-RNA 5'-UNTRANSLATED LEADER FUNCTIONS IN MITOCHONDRIAL TRANSLATION INITIATION AND TRANSLATIONAL ACTIVATION

We have used mutational and revertant analysis to study the elements o f the 54-nucleotide COX2 5'-untranslated leader involved in translatio n initiation in yeast mitochondria and in activation by the COX2 trans lational activator, Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5'-UTL by in, vitro mutagenesi s followed by mitochondrial transformation and gene replacement. The p henotypes of these mutants delimit a 31-nucleotide segment, from -16 t o -46, that contains several short sequence elements necessary for COX 2 5'-UTL function in translation. The sequences from -16 to -47 were s hown to be partially sufficient to promote translation in a foreign co ntext. Analysis of revertants of both the series of linker-scanning al leles and two short deletion/insertion alleles has refined the positio ns of several possible functional elements of the COX2 5'-untranslated leader, including a putative RNA stem-loop structure that functionall y interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5'-UTLs that is a potential rRNA bin ding site.