Mitochondrial gene expression at Castro, Italy.

Progress on the study of mitochondrial genes and their expression since the recent explosion of DNA sequence data was thoroughly reviewed this past June (22-27) at an EMBO Workshop on "Regulatory Mechanisms in Mitochondrial Gene Expression" held in southern Italy near Bari. Many interesting findings concerning transcription of mitochondrial genes and processing of their transripts were discussed. In addition, a host of fascinating new facts came to light, to be added to the lore that could perhaps best be termed the Natural History of Mitochondrial Genes. Mitochondrial transcription in mammalian cells and yeast is under intensive investigation. In Hela cell mitochondria, transcription of the H-strand (complementary to most transcripts) appears to start at either of two closely linked initiation points near the origin of H-strand DNA replication (G. Attardi, Cal. Tech.). The first products of this transcription are RNAs containing both the 12S and 16S rRNA sequences, some of which are rapidly processed to the mature molecules, while the others are poly-adenylated and relatively stable. Downstream genes are apparently transcribed as a result of read-through of a termination signal near the 3' end of the 16S rRNA gene. The possibility to analyze some of these events in vitro appears to be at hand, since R. A. Van Etten (Stanford) reported that mitochondrial RNA polymerase appears to initiate transcription specifically in the D-loop region as judged run-off transcription assays. J. C. Edwards (Chicago) reported studies on transcription of yeast mtDNA, in vivo and in vitro. By analyzing primary transcripts extracted from rhocells, labeled in vitro at their 5'-triphosphate ends, at least 17 different transcription initiation sites have been detected. (Transcription initiation in wild-type might differ from that in rho-s ' ) Experiments in vitro using purified yeast mitochondfial RNA polymerase and cloned fragments of yeast mtDNA as template revealed a clear specificity of initiation at promoters for the 21S and 16S rRNA genes as judged by run-off assays. In vitro deletion studies on the cloned DNAs are in progress to localize the sequences necessary for specific initiation: so far one can say that promoter function lies between -1 2 and +40 with respect to the initiation site. This was very nicely consistent with the report by H. TabakandK. Osinga(Amsterdam) that virtually the same 17bp sequence is found immediately upstream from both the 21S and 16S rRNA genes, strongly suggesting that it could be part of a promoter. Moreover they found 8bp of this sequence in front of the gene encoding the small rRNA ofKluyveromyces lactis mtDNA as well, further strengthening the contention that it is funtionally important. One of the highlights of the meeting was R. A. Butow's (Dallas) report that the prodigal gene, varl. has finally come home to mtDNA. The correct sequence of this region reveals an interesting gene indeed. For starters, it contains 90% A+T in a reading frame coding a protein of about 400 amino acid residues, showing that useful information can be encoded with very sparing use of Gs and Cs. In fact 40% of the G and C content of the gene is present in a GC-rich-cluster, similar to those that have been postulated to be regulatory of RNA processing sites, present in the reading frame and the mRNA. The genetic elements that determine the length of the various forms of varl polypeptide have now been identified as a second copy of the GC-rich-cluster (in inverted orientation) and runs of the Ash codon AAT of differing length. It appears possible from these findings that the GC-rich-clusters may behave as IS elements. A further dividend from this work is that a comparison of the gene sequence with the amino acid composition of the protein shows clearly that the codon ATA encodes Met in yeast mitochondria, as in mammalian mitochondria, but different from the standard Ile. Even the previous confusion about the location of the varl structural gene has paid off nicely. The