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Nucleic Acids Research Advance Access originally published online on March 28, 2007
Nucleic Acids Research 2007 35(7):2413-2427; doi:10.1093/nar/gkm159
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Nucleic Acids Research, 2007, Vol. 35, No. 7 2413-2427
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system

Paola Loguercio Polosa1, Stefania Deceglie1, Maria Falkenberg3, Marina Roberti1, Barbara Di Ponzio1, Maria Nicola Gadaleta1 and Palmiro Cantatore1,2,*

1Dipartimento di Biochimica e Biologia Molecolare "Ernesto Quagliariello", Università degli Studi di Bari, 2Istituto di Biomembrane e Bioenergetica, CNR, Via Orabona, 4, 70125 Bari, Italy and 3Department of Laboratory Medicine, Division of Metabolic Diseases, Karolinska Institutet, Novum, SE-141 86 Stockholm, Sweden

*To whom correspondence should be addressed. Tel: +39-080-5443378; Fax: +39-080-5443403; Email: p.cantatore{at}biologia.uniba.it

Received October 26, 2006. Revised March 1, 2007. Accepted March 2, 2007.

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.


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