(A and B) Frequencies of positively charged residues (R+K) that were shown to slow-down translation (Charneski and Hurst, 2013) in regions of programmed pauses. (A) Frequencies of R+K along the coding sequences in membrane (mem), cytosolic (cyt), and periplasmic (per) proteins. Membrane proteins are depleted of K and R in codons 16–60 (and also throughout the sequence) compared to cytosolic proteins. (B) Frequency of R+K in regions immediately preceding programmed pauses that occur in codons 16–60. Programmed pause events that occur in codons 16–60 were analyzed for each protein class separately, and the peptide sequences directly preceding pauses were aligned such that position 0 constitutes the pause codon position. The frequency of K+R in every position is shown as solid line. Dashed lines indicate the average frequencies of K+R in the entire protein sequences. No significant excess of positively charged amino acids preceding pauses is observed. (C) Codon rarity along coding sequence position, as indicated by mean relative synonymous codon usage (RSCU) (Sharp et al., 1986). Most of the bias for rare codons occurs before codon 15 (dashed line), while the pause-enrichment in membrane proteins occurs only later. (D) Frequencies of G (Gly) residues, whose G-nucleotide-rich codons may mediate pause (Li et al., 2012), along the coding sequences. Membrane proteins show only a slight elevation of Gly occurrence, which is not specific to the region of codons 16–60. (E) Analysis of coding reading frames of SD-like sequences preceding pauses indicates that most such motifs do not occur in frames that would encode glycines. Left: the canonical SD sequence (red) and definition of frames. Frame 1 was defined as the frame in which the canonical SD encodes Gly–Gly dipeptide. Right: distribution of pause-preceding SD motifs in each frame. The analysis was done separately for membrane proteins in the codon range 16–60, for the other codons in membrane proteins, or for cytosolic proteins.