Inverse toeprinting: a new tool to decipher the translational arrest code

While it has become increasingly apparent that the amino acid sequence of a nascent polypeptide can modulate its rate of synthesis, tools to decipher the underlying translational arrest code are still lacking. Here, we present inverse toeprinting, a new method to map the position of bacterial ribosomes arrested on messenger RNAs during in vitro translation. Unlike the widely used ribosome profiling approach, our method protects the entire coding region upstream of a stalled ribosome, making it possible to work with transcripts of unknown sequence. To demonstrate this, we use inverse toeprinting to characterize the pausing landscape of free and drug-bound ribosomes engaged in translation of a random transcript library. The arrest motifs that we identified correlate well with previous in vivo profiling data. Inverse toeprinting also allows us to determine the pause strength of individual motifs and thus provides a means to decipher the bacterial arrest code that can be adapted to other sequence-dependent translational processes.