Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system.
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.
Polosa
Paola Loguercio
PL
Dipartimento di Biochimica e Biologia Molecolare Ernesto Quagliariello, Università degli Studi di Bari, Istituto di Biomembrane e Bioenergetica, CNR, Bari, Italy.
Deceglie
Stefania
S
Falkenberg
Maria
M
Roberti
Marina
M
Di Ponzio
Barbara
B
Gadaleta
Maria Nicola
MN
Cantatore
Palmiro
P
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D047328:Paracentrotus / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D018411:Spodoptera / Q000166:cytology; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C119291:mitochondrial D-loop-binding protein, Paracentrotus lividus; D012321:DNA-Directed RNA Polymerases