Figure 3.

The role of Rbs1 in controlling Pol III assembly is supported by the 3′ regulatory region of the RPB10 gene. The modified versions of the RPB10 gene that lacks designated sequences in the 5′ and/or 3′ regulatory regions were constructed on the plasmids as described in Supplementary data. (A) Effect of deletions in the 3′ UTR on suppression of the rpc128-1007 Pol III assembly mutant by RPB10. Δ3′ 154, Δ3′ 231 and Δ3′ 253, respectively, limited 3′UTR to 154, 231 and 253 nucleotides downstream a stop codon. The control strain (wt) and transformants of the rpc128-1007 mutant with derivatives of [RPB10] containing designated deletions, the [RPB10] control plasmid, and the empty vector [–] were grown on SC-ura plates, replicated on YPD plates, and incubated for 3 days at the respective temperatures. (B) RNA isolated from transformants of the rpc128-1007 mutant with derivatives of [RPB10] containing designated deletions and the [RPB10] control plasmid was used to synthesis of cDNA samples that were analyzed by RT-qPCR. mRNA levels were normalized to ACT1 mRNA and calculated relative to amounts in the strain harboring the [RPB10] control plasmid, which was set to 1. Bars represent the mean ± standard deviation of three independent experiments. P values were calculated using a two-tailed t-test. (C) Δ3′154 deletion in 3′ UTR of RPB10 negatively affected suppression of the Pol III assembly mutant rpc128-1007 by RBS1. A double rpb10Δ rpc128-1007 mutant that harbored the [RPB10 Δ3′154] plasmid was additionally transformed with the [RBS1] plasmid or empty vector [–]. A double rpb10Δ rpc128-1007 mutant that harbored [RPB10], a single rpc128-1007 mutant that harbored [RBS1], and the wild type strain (wt) were additionally transformed with the respective empty vectors and served as controls. Yeast cells that were grown on an SC-ura-leu plate were replicated on YPD plates and incubated for 3 days at the respective temperatures.