ABSTRACT
Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms.
Subject(s)
DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Mitochondria/enzymology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Paracentrotus/genetics , Transcription, Genetic , Amino Acid Sequence , Animals , Baculoviridae/genetics , Cell Line , Cloning, Molecular , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/chemistry , Gene Expression Regulation , Mitochondria/genetics , Mitochondrial Proteins/chemistry , Molecular Sequence Data , Paracentrotus/enzymology , Sequence Alignment , Spodoptera/cytologyABSTRACT
The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding.
