<p>(A) The transcription start site (TSS) and translation initiation site are the same in leaderless genes. No 5’ UTR and no Shine-Dalgarno (SD) sequence imply that an assembled 70S ribosome engages the 5’ terminal initiation codon directly, followed by elongation to translate the ORF. Individual sequence reads from RNA-seq (green) and ribosome profiling (Ribo-seq, orange) analyses were mapped to the genome, and the abundance of the individual reads is indicated by the height of the peaks. In leaderless translation RNA-seq and ribosome profiling have coincident 5’ boundaries. The 5’ triplet is nearly always ATG or GTG and, in the typical example shown, corresponds to the predicted N-terminus of the annotated ORF. (B) Traditional gene structures generate nested ribosome profiling profiles, with a 5’ UTR that includes an SD ribosome-binding site upstream of the initiating methionine codon (ATG). The 30S and 50S ribosomal subunits assemble at the SD to form a complete 70S ribosome that begins translation at the adjacent AUG with an N-terminal formylated methionine (fM) amino acid residue. RNA-seq reads (green) indicate positive-strand transcription at this locus, and upstream of an annotated ORF. Mapped ribosome profiling reads (orange) begin downstream of the onset of RNA-seq reads, and ~17–35 nt upstream of the initiation codon of the annotated ORF. In both examples, JCVI correctly predicted the respective ORFs (black).</p
