%0 Journal Article
%T Functional suppression of a yeast maf1 deletion mutant by overdose of the N-terminal fragment of the largest RNA polymerase III subunit, C160.
%A Łopusińska A
%A Farhat M
%A Cieśla M
%J Gene
%V 930
%N 0
%D 2024 Dec 20
%M 39142551
%F 3.913
%R 10.1016/j.gene.2024.148839
%X Maf1 is a general and global negative regulator of RNA polymerase III (Pol III) transcription. Under repressive conditions, Maf1 binds directly to the Pol III complex and sequesters Pol III elements that are involved in transcription initiation. To further understand Pol III regulation, we searched for genetic bypass suppressors of a maf1 deletion mutant (maf1Δ) of Saccharomyces cerevisiae. Strains that carried maf1Δ were temperature-sensitive on media that contained nonfermentable carbon sources and showed the antisuppressor phenotype. Suppressors allowed colonies to grow at the restrictive temperature on glycerol media and partially complemented the antisuppressor phenotype of maf1Δ. DNA plasmids that were identified as overdose suppressors encoded N-terminal fragments of the largest Pol III subunit, C160 of various lengths. The shortest fragment, 372 amino acids long, the overdose of which partially complemented the antisuppressor phenotype and temperature-sensitive respiratory growth of maf1Δ, was named C160-NTF. In this study, we showed that the expression of HA-tagged C160-NTF resulted in accumulation of approximately 40 kDa protein that was distributed throughout the yeast cell, in the cytoplasm and nucleus. The overdose of C160-NTF led to decrease of tRNA transcription in maf1Δ mutant cells, demonstrating functional suppression. Levels of newly synthesized individual tRNAs and Pol III occupancies on tRNA genes were decreased by C160-NTF in the maf1Δ mutant. Additionally, we analyzed the effect of C160-NTF overproduction and the presence of Maf1 on the associations among Pol III subunits. Previous structural analyzes of Pol III have indicated that the N-terminal region of C160 interacts with the C82-34-31 heterotrimeric Pol III subcomplex. We suggest that the negative effect of C160-NTF overdose on tRNA transcription is related to defective Pol III assembly, because overproduction of C160-NTF altered C160 interactions with C34 and C82 in the maf1Δ mutant.