Point mutation in C128 subunit of the yeast RNA polymerase III compensates deffect in regulation of the tRNA biosynthesis in the absence of Maf1.
Malgorzata Ciesla, Karol Balicki, Magdalena Boguta
Genetics, IBB PAS, Pawinskiego 5A, Warsaw, 02-106, Poland
Maf1 is a repressor of RNA polymerase III (pol III) in yeast. Cells with inactivated Maf1 have elevated tRNA levels and show temperature sensitive growth defect. Suppressor of maf1 deletion was isolated as a mutation with a cold sensitive phenotype (cs). To clone the gene that complements the cs phenotype, cells were transformed with yeast genomic libraries on a single copy or a multicopy plasmid. Several genomic fragments were selected, identified by sequencing and subcloned. According to genetic analysis, a RET1 gene was one linked to the cs mutation, referred to as ret1-119. RET1 encodes the second largest subunit of pol III, C128. Sequencing of RET1 mutated gene identified a single nucleotide substitution leading to change of the conserved glycine 1007 in C128 protein. The RPB10 gene encoding a conserved subunit common to all three RNA polymerases was cloned as a suppressor of ret1-119 using single copy gene library. Four independent inserts that complemented the cs phenotype of ret1-119 selected from the multicopy library contained the RBS1 gene and one insert contained the PRT1 gene. Both RBS1 and PRT1 encode RNA-binding proteins. The product of PRT1 functions in translation, whereas the role of RBS1 remains unknown. The ret1-119 mutant showed a decrease in tRNA levels in comparison with the control. Both the RPB10 and RBS1 genes on a plasmid raised the tRNA levels when introduced into ret1-119 cells. This result points to Rbs1 protein as a potential pol III activator.