FIG. 4.

In vivo transcriptional activity of mutant M50 mga alleles expressed from a constitutive PrpsL promoter. (A) GusA activity of whole-cell lysates (top). Production of β-glucuronidase activity was determined for lysates from an mga-deleted Pmrp-gusA reporter strain KSM149 containing plasmids expressing mga alleles from the PrpsL promoter (M4 Mga [lane 1], M50 Mga [lane 2], vector only [lane 3], and M50 Mga mutants [S26N [lane 4], P361A [lane 5], R461M [lane 6], and S26N/R461M [lane 7]). GusA units represents a measure of absorbance (A₄₂₀)/protein concentration (μg/ml), and values are the averages of at least three independent experiments. The percent activity compared to M4 Mga is indicated above each bar. Western analysis was performed on whole-cell lysates using both an anti-His antibody for Mga-His protein levels (middle) and anti-Hsp60 antibodies as a control for loading (bottom). WT, wild type. (B) Northern analysis of Mga-specific transcriptional activation. Transcript levels for the Mga-regulated gene mrp were determined using total RNA (5 μg) isolated from an mga-inactivated M22 strain, AL168-mga, containing plasmids expressing Mga alleles from the PrpsL promoter (M4 Mga [lane 1], M50 Mga [lane 2], vector only [lane 3], and M50 Mga mutants S26N [lane 4], P361A [lane 5], R461M [lane 6], and S26N/R461M [lane 7]). Blots were stripped and reprobed with 23S rRNA to serve as a loading control (directly below). Western analysis was performed on whole-cell lysates using both an anti-His antibody for Mga-His protein levels (third panel from the top) and with antibodies to Hsp60 as a control for loading (bottom). All blots shown are representative of data from three independent experiments. (C) Slot blot analysis of mga transcripts produced from the constitutive PrpsL promoter. Total RNA (1 μg) was isolated from the mga-inactivated M22 strains above and probed for M50 mga (left) and 23S rRNA as a loading control (right).