Biochemical and genetic dissection of the Saccharomyces cerevisiae RNA polymerase C53 subunit through the analysis of a mitochondrially mis-sorted mutant construct.
RPC53 has previously been shown to encode an essential subunit required for tRNA gene transcription by RNA polymerase C in vivo (Mann, C., Micouin, J.-Y., Chiannilkulchai, N., Treich, I., Buhler, J.-M., and Sentenac, A. (1992) Mol. Cell. Biol. 12, in press). In this paper, we have determined that an unusual rho+ lethality associated with the rpc53::HIS3-1 disruption mutation is due to the inadvertent formation of a Pet56-C53 fusion protein. This fusion protein is missorted to mitochondria, thereby reducing the quantity of the C53 subunit available for RNA polymerase C assembly. We show that the carboxyl-terminal region of C53 contains the essential functional domain of the subunit and that a mutant RNA polymerase containing only this domain is thermolabile for its function in vivo and in vitro. The thermolability of the carboxyl-terminal C53 domain is suppressed by five different genes on multicopy plasmids, including RPC160, encoding the largest subunit of RNA polymerase C and SSD1/SRK1, which has been implicated in the activity of protein phosphatases.
Chiannilkulchai
N
N
Service de Biochimie et Génétique Moléculaire, Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
Moenne
A
A
Sentenac
A
A
Mann
C
C
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005800:Genes, Fungal; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D015698:Genomic Library; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012320:RNA Polymerase III / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012234:Rho Factor / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017384:Sequence Deletion; D014158:Transcription, Genetic
D004271:DNA, Fungal; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012234:Rho Factor; D012343:RNA, Transfer; D012320:RNA Polymerase III