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The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions
Author(s) -
FernandezSilva Patricio,
MartinezAzorin Francisco,
Micol Vicente,
Attardi Giuseppe
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.5.1066
Subject(s) - biology , leucine zipper , dna , mitochondrial dna , intramolecular force , dna binding protein , transcription factor , leucine , biophysics , transcription (linguistics) , genetics , microbiology and biotechnology , gene , stereochemistry , amino acid , linguistics , philosophy , chemistry
The human mitochondrial transcription termination factor (mTERF) cDNA has been cloned and expressed in vitro , and two alternative precursors of the protein have been imported into isolated mitochondria and processed to the mature protein. The precursors contain a mitochondrial targeting sequence, and the mature mTERF (342 residues) exhibits three leucine zippers, of which one is bipartite, and two widely spaced basic domains. The in vitro synthesized mature protein has the expected specific binding capacity for a double‐stranded oligonucleotide containing the tridecamer sequence required for directing termination, and produces a DNase I footprint very similar to that produced by the natural protein. However, in contrast to the latter, it lacks transcription termination‐promoting activity in an in vitro system, pointing to another component(s) being required for making mTERF termination‐competent. A detailed structure–function analysis of the recombinant protein and mutagenized versions of it by band shift assays has demonstrated that both basic domains and the three leucine zipper motifs are necessary for DNA binding. Furthermore, a variety of tests have shown that both the recombinant and the natural mTERF bind to DNA as a monomer, arguing against a dimerization role for the leucine zippers, and rather pointing, together with the results of mutagenesis experiments, to intramolecular leucine zipper interactions being required to bring the two basic domains in close register with the mTERF target DNA sequence.