Ordered processing of the polygenic transcripts from a mitochondrial transfer-RNA gene cluster in K. lactis.

In Saccharomyces cerevisiae, transcription of the mitochondrial genome starts at multiple initiation sites and is followed by the processing of multigenic transcripts at the 5' and 3' termini of tRNA sequences and in some intergenic regions. We have used a comparative approach to investigate the structure and function of the latter processing sites. We present here an analysis of the transcripts of a cluster of tRNA genes from the mitochondrial genome of Kluyveromyces lactis. The gene order of this cluster is the same as that of the cluster in S. cerevisiae but the sequence of the intergenic regions is different. A detailed analysis of transcripts has been performed using S1 mapping and primer extension techniques. The results can be summarized as follows: (1) transcription of the cluster very probably starts at initiation sites having the nonanucleotide sequence TTATAAGTA (which acts as a promoter in S. cerevisiae) and yields polygenic transcripts; (2) processing of these transcripts seems to occur through an ordered pathway of endonucleolytic events in which some tRNA sequences are preferentially excised and some endonucleolytic cuts occur more readily than others; (3) in two intergenic regions, strong signals indicate the existence of processing events. The sequences around these sites are similar in sequence and localization to S. cerevisiae intergenic processing sites, indicating a possible functional importance in maintaining a conserved order of tRNA genes in different species of yeasts.