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Isolated rat liver mitochondria in the presence of an appropriate incubation buffer are able to support DNA transcription and RNA processing in a way qualitatively and quantitatively similar to that used by intact cells. This system is also able to synthesize an RNA species of 155-175 nucleotides which probably corresponds to the 7S RNA, a type of RNA found so far only in growing cells. The role of this RNA in the mitochondrial replication and transcription processes, in relation to the cell metabolism, is discussed. [ABSTRACT FROM AUTHOR]

We have determined the base sequence of the restriction fragment Bam1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene cluster, between the tRNA and tRNA genes, is a sequence of 134 bp which constitutes the only non-coding region of this DNA so far identified. The distinctive organization of the tRNA genes and the extreme size reduction of the non-coding region suggest the existence of unique mechanisms for the regulation of gene expression in this organism. [ABSTRACT FROM AUTHOR]

Footprinting studies with the purine-modifying agent dimethyl sulphate were performed in Paracentrotus lividus eggs and embryos to analyze in vivo the interactions between protein and mitochondrial DNA. Footprinting in the small non-coding region and at the boundary between the ND5 and ND6 genes revealed two strong contact sites corresponding with the in vitro binding sequences of mitochondrial D-loop-Binding Protein (mtDBP). The analysis of the pause region of mtDNA replication showed a strong footprint corresponding with the binding site of the mitochondrial Pause region-Binding Protein-2 (mtPBP-2), but only a very weak signal at the binding site of the mitochondrial Pause region-Binding Protein-1 (mtPBP-1), which in vitro binds DNA with high efficiency. In vitro and in vivo analysis of the 3' end-region of the two rRNA genes showed no significant protein-DNA interactions, suggesting that, in contrast to mammals, the 3' ends of sea urchin mitochondrial rRNAs are not generated by a protein-dependent transcription termination event. These and other data support a model in which expression of mitochondrial genes in sea urchins is regulated post-transcriptionally. Footprinting at the five AT-rich consensus regions allowed the detection of a binding site in the non-coding region for an as-yet unidentified protein, mtAT-1BP. The occupancy of this site appears to be developmentally regulated, being detectable in the pluteus larval stage, but not in unfertilized eggs.

A mitochondrial protein, able to specifically bind two double-stranded homologous sequences of sea-urchin mitochondrial DNA, has been partially purified from Paracentrotus lividus eggs. This protein, present at a low concentration, is a polypeptide of 40 kDa. One of the binding sequences, located in the main non-coding region, contains the replication origin of the mitochondrial DNA H-strand. By a combination of band-shift, DNase footprinting, and modification interference analyses with homologous and heterologous probes we identified YCYYATCAN(A/T)RC as the minimum sequence required for the binding. The protein also shows a single-stranded DNA-binding activity, as it is able to specifically interact with one of the strands of the binding sites. These features are consistent with a function of the protein in the modulation of sea-urchin mitochondrial DNA replication during the development stages.

This paper reports the mapping of both mature and precursor Paracentrotus lividus mitochondrial transcripts. Several mtRNAs were found to have 5' and 3' termini which differ from those inferred through DNA sequencing (Cantatore et al. 1989). The 3' ends of the two rRNAs (12S and 16S) overlap with the downstream transcripts (tRNAGlu and CoI mRNA) by 5 and 10 nt respectively. The 132 nt non-coding region is extensively transcribed: in particular it contains a 124 nt RNA and the 5' end of a possible precursor of 13 clustered tRNAs. This latter overlaps by 7 nt with the 3' end of the 124 nt RNA. In addition to the mature RNAs, 32 high molecular weight RNAs, which are probably the precursors of the smaller more abundant mature species, were detected by Northern blotting. The mapping of these transcripts indicates that they are processed at the level of tRNA or tRNA-like sequences and suggests the existence of two transcription initiation sites upstream of the ND1 and the cytochrome b genes respectively. In the light of these results it appears that P. lividus mitochondrial DNA transcription takes place via multiple and probably overlapping transcription units. Moreover, the wide variation in the steady-state levels of the mature mRNAs indicates that sea urchin mitochondrial DNA expression is also regulated at the level of RNA decay.