Figure 5. Opposite effects have been observed in HXT1 promoter activity in strains with altered RNAP III.

(A) Schematic representation of glucose uptake and phosphorylation in yeast cells. Hxt, hexose transporter; P, phosphorylation; Hxk1, hexokinase 1; Hxk2, hexokinase 2; Glk1, glucokinase 1. WT, maf1Δ, rpc128-1007 yeast cells and single or double HXK1, HXK2, GLK1 knockouts strains in WT, maf1Δ and rpc128-1007 genetic background were cultured in YPD (C) or YPGly (D) rich medium under either inducing (2% glucose) or repressing (2% glycerol) conditions. Maf1 deficiency increases HXT1 expression (B) on glucose and Hxk2 activity regardless of carbon source (C, D). Metabolic effects observed in rpc128-1007 correlate with decreased HXT1 expression (B) and decreased hexokinase activity in glucose-rich medium (C), but increased hexokinase activity in glycerol rich medium (D). Compromised RNAP III and maf1Δ have an effect on enzymes in lower glycolysis: Tdh1-3 and Cdc19 activities (E, F). The WT strain (MB159-4D), maf1Δ and rpc128-1007 mutant strains were grown under 2% glucose and 2% glycerol conditions. The experiment was performed in cell-free extracts isolated from the aforementioned strains. Data are expressed as the mean obtained from at least three independent experiments measured in triplicate. The standard deviations are shown. Enzymatic assays were performed in cell-free extracts. The reaction rates were monitored by measuring NADH concentration change over time at 340 nm. V_max mean value is expressed as µmol·min⁻¹·mg⁻¹ protein (C, D, E, F). (B) HXT1 expression was measured in WT [pBM2636], maf1Δ [pBM2636] and rpc128-1007 [pBM2636] strains by using the lacZ reporter gene system [66]. β-galactosidase activity was assayed in cell-free extracts. The error bars indicate the standard deviation from three independent transformants assayed in triplicate. Asterisk (*) indicate P-value <0.05 and double asterisk (**) illustrate P-values <0.1 according to Student's t-test averaged from all technical repeats.