Extended Data Fig. 1. Additional characterization of ribosome stalling in vitro.

(A) A second example of nascent chain products resulting from in vitro translation of iterated AAG or AAA lysine codons in human cell lysate, as in Fig. 1A. The positions of nascent chain products containing 4, 9, or 12 lysines are indicated. (B) Analysis of iterated AAG versus AAA codons for stalling in rabbit reticulocyte lysate. The translation reaction was performed for 20 min after which the proportion of stalled products was assessed by the relative amounts of peptidyl-tRNA versus full length polypeptide. The ‘background’ of ~20% peptidyl-tRNA even in the absence of stalling is due to failed termination at the stop codon, which is located within a few nucleotides of the 3’ end of the mRNA. Later in vitro stalling experiments with a longer 3’UTR that protrudes outside the mRNA channel showed improved termination efficiency (~95%). An overly short 3′UTR presumably makes the mRNA more flexible in the mRNA channel and less able to recruit eRF1. Multiple experiments such as this one were quantified to produce the graph shown in Fig. 1B. (C) Time course of the appearance of full length (FL) product for constructs containing the indicated number of iterated AAG or AAA codons. Translation was synchronised by first pausing the ribosome at a run of rare leucine codons just preceding the poly-basic encoding sequence, then restarting translation at time 0 by addition of tRNA. The mean ± SEM for each time point calculated from two experiments are plotted.