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The promiscuous streptococcal plasmid pLS1 encodes for the 5.1 kDa RepA protein, involved in the regulation of the plasmid copy number. Synthesis of RepA was observed both in Bacillus subtilis minicells and in an Escherichia coli expression system. From this system, the protein has been purified and it appears to be a dimer of identical subunits. The amino acid sequence of RepA has been determined. RepA shows the alpha helix-turn-alpha helix motif typical of many DNA-binding proteins and it shares homology with a number of repressors, specially with the TrfB repressor encoded by the broad-host-range plasmid RK2. DNase I footprinting revealed that the RepA target is located in the region of the promoter for the repA and repB genes. Trans-complementation analysis showed that in vivo, RepA behaves as a repressor by regulating the plasmid copy number. We propose that the regulatory role of RepA is by limitation of the synthesis of the initiator protein RepB.

Proteins of the small and large subunits of mitcchondrial ribosomes from the yeast Succharomyces cerevisiae were isolated and characterized by two-dimensional gel electrophoresis. Ribosomal proteins of the large subunit were separated by reverse-phase HPLC and up to 37 amino acid residues of the N-terminal sequences of L3, L4, L9 and L31 were determined. No significant homology to ribosomal protein sequences so far determined from other organisms was found.

We have determined the N-termini of 26 proteins of the large ribosomal subunit from yeast mitochond~a by direct amino acid micro-sequencing.The N-terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r-) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N-termini of the mature proteins, the putative leader peptides eesponsible for mitochondrial matrix-targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found. a programme for the isoiation and sequencing of mitochondrial r-proteins 121. N-Terminal sequence data were used for cloning of the nuclear genes by oligonucleotide hybridisation [3,4]. In this way several nuclear genes coding for mitochondrial r-proteins have been cloned and anaIysed [3-71, and it was found that 3 of the sequences are significantly related to eubacterial r-proteins. More generally, yeast nuclear genes coding for mitochondrial proteins were cloned by genetic complementation of pet mutants (for review see [8]). Pet mutant genes often affect gene products that are either directly involved in the oxidative metabolism of mitochondria or are necessary for expression of its activity [9]. For example, the PET-genes MRPl and MRPZ were found to code for mitochondria1 r-proteins [IO]. It is to be expected that several more MRPs will be analysed, since most of the mitochondrial r-proteins are essential for the translational activity of mito-ribosomes [4-7,10,1 I]. Recently, McMullin et al. [ll] identified PET123 as a component of the small ribosomai subunit in yeast. Nevertheless, in most cases it is difficult to define the exact function of the PET-genes, and additional biochemical analysis is necessary to assign them a function. Even with more data this function might remain unknown [12]. Therefore, N-terminal sequences of mitochondrial r-proteins could be a fruitful basis not only for cloning of the corresponding genes, but also for the identification and/or assignment of other PET-gene products in the future.In the present study we show the results of extended N-terminal amine acid sequence analysis of 26 mitochondrial r-proteins, Proteins MRP7, YMR-26 and 51

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