Nam1p, a Protein Involved in RNA Processing and Translation, Is Coupled to Transcription through an Interaction with Yeast Mitochondrial RNA Polymerase

• Published in: The Journal of biological chemistry 2001-03, Vol.276 (11), p.8616-8622
• Main Authors: Rodeheffer, MatthewS, Boone, BradenE, Bryan, AnthonyC, Shadel, GeraldS
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Conserved Sequence; COXI gene; CYTB gene; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - metabolism; Mitochondrial Proteins; Molecular Sequence Data; Nam1 protein; Repetitive Sequences, Amino Acid; RNA - metabolism; RNA, Mitochondrial; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Temperature; Transcription Factors - physiology; Transcription, Genetic; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M009901200

Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain. Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain. The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Δ2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p. In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTBand COXI transcripts, which is a primary defect observed inNAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obviousCOX1/CYTB transcript defect. This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes. These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery.