Time course of ribosome clearance following lincomycin treatment in light <i>versus</i> dark.

<p>(A) Experimental design. Lincomycin (LIN) inhibits peptide bond formation only when the nascent peptide is shorter than approximately five amino acids. Therefore, ribosomes with longer nascent peptides continue to elongate in its presence. LIN was applied to seedlings at midday in the light or after 30 minutes of dark adaptation; this period of dark adaptation is sufficient for ribosome occupancy on <i>psbA</i> mRNA to decrease to its dark steady-state level (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007555#pgen.1007555.g005" target="_blank">Fig 5</a>). Material was harvested just prior to LIN treatment, and 12 or 30 minutes later. (B) Distribution of ribosomes along four chloroplast ORFs following LIN treatment in the dark or light. The plots show the normalized abundance of ribosome footprints with 3’ ends at each position. Plots for each of two replicates are shown separately to illustrate reproducibility. The region occupied by initiating ribosomes (first 7 codons) is shaded in gray. (C) Fractional change in ribosome footprint abundance on each chloroplast ORF following LIN treatment in the light and dark. The apical half of leaves 2 and 3 were processed for ribosome profiling. Each line shows the percent of the initial RPKM for a single chloroplast ORF over time following LIN treatment. The data from two replicate datasets are shown in separate plots to illustrate trends and variation. Footprints mapping to the first seven codons of each ORF were excluded from read counts because LIN inhibits these ribosomes. Ribosomes that build up at specific ORF-internal sites following LIN treatment (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007555#pgen.1007555.g007" target="_blank">Fig 7</a>) were also excluded based on the criteria and rationale explained in Materials and Methods. Only genes whose average RPKM was greater than 100 at t = 0 are included (n = 70).