Figure 4: Altered translation of poxB is nascent peptide dependent.
(a) Toeprinting analysis of distribution of translating unmethylated (WT) or methylated (M) ribosomes along the segment of the poxB mRNA containing the codon Asn118. The sequences of the mRNA templates encoding the wild-type PoxB (lanes 1 and 2) or the frameshift mutant PoxB-fs (lanes 3 and 4), with the altered sequence of amino-acid residues 102–117 (amino-acid residues in green), are shown on the right with the green arrows indicating the single-nucleotide deletion and insertion used to generate the mutations. The intensity of the toeprint band at the poxB nucleotide G368 (an arrow and a dot on the gel) reflects the occupancy of the codon Asn118 (boxed in red in the sequences). (b) Quantification of the differential pausing (differential intensity of the toeprint band) at G368 of poxB or poxB-fs of methylated over unmethylated ribosomes. For comparison, differential intensities of bands of the 10 nucleotides preceding and following position G368 were also calculated after the background intensity was subtracted. The integrated density of five consecutive bands with seemingly similar intensities was used to normalize the data to account for potential difference in loading. Mean values were originated from three independent experiments; error bars represent the s.d. of the mean.
