No abstract

The protein specified by the Saccharomyces cerevisiae nuclear gene PET111 specifically activates translation of the mitochondrially coded mRNA for cytochrome c oxidase subunit II (Cox2p). We found Pet111p specifically in mitochondria of both wild-type cells and cells expressing a chromosomal gene for a functional epitopetagged form of Pet111p. Pet111p was associated with mitochondrial membranes and was highly resistant to extraction with alkaline carbonate. Pet111p was protected from proteolytic digestion by the mitochondrial inner membrane. Thus, it is exposed only on the matrix side, where it could participate directly in organellar translation and localize Cox2p synthesis by virtue of its functional interaction with the COX2 mRNA 5-untranslated leader. We also found that Pet111p is present at levels limiting the synthesis of Cox2p by examining the effect of altered PET111 gene dosage in the nucleus on expression of a reporter gene, cox2::ARG8 m , that was inserted into mitochondrial DNA. The level of the reporter protein, Arg8p, was one-half that of wild type in a diploid strain heterozygous for a pet111 deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111 on a high-copy plasmid. Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.

Cell-based assays can be used to identify molecules that are selectively toxic to cells with a predetermined genetic background, including mutations in genes involved in the cell cycle and its checkpoints, for which there are currently no selectively toxic compounds.

It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease.

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.