Evidence that hALT contributes to 30S biogenesis. (A,B) Polysome analysis of Δ7 prrn strains (as indicated) without (A) or with (B) chloramphenicol treatment before lysis. Shown are representative A254 traces of sucrose gradients, with peaks corresponding to subunits (30S, 50S), monosomes (70S), and polysomes (Polys) indicated. The bar graphs below show the quantification of 30S, 50S, 70S, and polysomes (as indicated) in control (gray) and mutant (red, U1532A/C1533G; blue, U1532C/C1533U) strains. Data represent the mean ± SEM from at least three biological replicates. A two-tailed t-test was used to evaluate differences from the WT. Uncorrected P-values: (*) P < 0.05; (**) P < 0.005. (C) Analysis of precursor 17S rRNA in the control and mutant strains. RNA was extracted from various fractions of sucrose gradients from experiments like those of A and analyzed by PAGE. A representative gel, analyzing RNA of the control strain, is shown, with specific fractions indicated. Arrowheads denote bands corresponding to precursor (17S) and mature (23S, 16S) rRNAs. For the bar graph below, 17S and 16S rRNA bands were quantified and used to calculate the proportion 17S (17S/[17S + 16S]) within each fraction from control (gray) and mutant (red, U1532A/C1533G; blue, U1532C/C1533U) cells. Data represents the mean ± SEM of three biological replicates. A two-tailed t-test was used to evaluate differences from the WT. Uncorrected P-value: (*) P < 0.05.
