Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase.
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 (rpo41Delta2), 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 CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/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.
Rodeheffer
M S
MS
Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322, USA.
Boone
B E
BE
Bryan
A C
AC
Shadel
G S
GS
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
C063626:MTF2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D000077278:RNA, Mitochondrial; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012313:RNA; D012321:DNA-Directed RNA Polymerases