A mutation in the yeast mitochondrial core RNA polymerase, Rpo41, confers defects in both specificity factor interaction and promoter utilization.
The yeast mitochondrial RNA polymerase (RNAP) is composed of the core RNAP, Rpo41, and the mitochondrial transcription factor, Mtf1. Both are required for mitochondrial transcription, but how the two proteins interact to create a functional, promoter-selective holoenzyme is still unknown. Rpo41 is similar to the single polypeptide bacteriophage T7RNAP, which does not require additional factors for promoter-selective initiation but whose activity is modulated during infection by association with T7 lysozyme. In this study we used the co-crystal structure of T7RNAP and T7 lysozyme as a model to define a potential Mtf1 interaction surface on Rpo41, making site-directed mutations in Rpo41 at positions predicted to reside at the same location as the T7RNAP/T7 lysozyme interface. We identified Rpo41 mutant E1224A as having reduced interactions with Mtf1 in a two-hybrid assay and a temperature-sensitive petite phenotype in vivo. Although the E1224A mutant has full activity in a non-selective in vitro transcription assay, it is temperature-sensitive for selective transcription from linear DNA templates containing the 14S rRNA, COX2, and tRNAcys mitochondrial promoters. The tRNAcys promoter defect can be rescued by template supercoiling but not by addition of a dinucleotide primer. The fact that mutation of Rpo41 results in selective transcription defects indicates that the core RNAP, like T7RNAP, plays an important role in promoter utilization.
Matsunaga
Michio
M
Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA.
Jaehning
Judith A
JA
D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000502:physiology*; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000502:physiology; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D014764:Viral Proteins
D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009238:N-Acetylmuramoyl-L-alanine Amidase