Maria C. Costanzo

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

ABSTRACT Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5′ untranslated leaders (5′-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5′-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5′-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5′-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.

Three yeast proteome databases: YPD, pombePD, and CalPD (MycopathPD)

Publisher Summary Proteomes fungal databases, YPD (for Saccharomyces cerevisiae), PombePD (for Schizosaccharomyces pombe), and CalPD (for Candida albicans), work together to present a comprehensive view of the molecular and cell biology of the genes and proteins of the fungal world. YPD, CalPD, and PombePD are part of a group of databases collectively called the BioKnowledge Library, which also includes databases for complex organisms, such as the worm Caenorhabditis elegans (WormPD), a model for development of a multicellular organism, and a protein survey database for humans. This chapter we provide detailed coverage of the YPD database, which has served as the prototype for the creation of all other volumes of the BioKnowledge Library. The chapter presents the unique features of the newer fungal databases PombePD and CalPD. CalPD is expected to be incorporated into MycoPathPD, a new volume that will include genes and proteins of C. albicans and other human pathogenic fungi. YPD is recommended as a good initiation site for newcomers.

A point mutation in the 5′-untranslated leader that affects translational activation of the mitochondrial COX 3 mRNA

The 613-base 5′-untranslated leader (5′-UTL) of the Saccharomyces cerevisiae mitochondrial COX 3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox 3 point mutations, using a set of defined COX 3 deletions, and found one to be located in the region coding the 5′-UTL. The strain carrying this allele was specifically defective in translation of the COX 3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5′-UTL (T inserted between bases-428 and-427 with respect to the start of translation) and a G to A substitution at+3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox 3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5′-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5′-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet 54, one of the COX 3-specific translational-activator genes. This phenotypic enhancement suggests that the-400 region of the 5′-UTL, where the mutation is located, is important for Pet54p-COX 3 mRNA interaction.

Primary structure of wild-type and mutant alleles of the PET494 gene of Saccharomyces cerevisiae

SummaryThe product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase. We have determined the DNA sequence of a 1.9 kb fragment carrying PET494. The sequence contains a single long open reading frame of 489 codons. This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon. The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5′ ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame. The predicted PET494 protein has a highly basic amino-terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic. The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic. The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function. A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations liked to PET494.