(A) Heatmap of ribosome density downstream of Ser codons in samples harvested by filtration using lysis buffers containing Cm, 150 mM MgCl2, or 1 M NaCl (L26, L27, and L28, respectively). (B) Model of how pausing affects ribosome density. Downstream of a pause site (shown in red), ribosomes continue elongation and are released at stop codons, such that downstream density drops until a new steady state is reached. (C) Heatmap of ribosome density downstream of Ile codons in untreated cells (L29) and after 10 min of mupirocin treatment (L30). (D) Schematic of the method used for panels 6E and 6F: from a single culture, samples were harvested by rapid filtration or by directly freezing the culture. Ribosomes were then pelleted through a sucrose cushion. (E) Plots and heatmaps of average ribosome density aligned at Ser codons in untreated cells that were filtered (L29) or frozen (L33). (F) Northern blot of Ser, Gly, and Ile tRNAs after periodate oxidation and β-elimination, a treatment that removes the final nt of tRNAs that are not charged. As a negative control, an aliquot of tRNA from filtered or frozen samples were pretreated in alkaline conditions to deacylate tRNA.