Active ribosome profiling with RiboLace

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information concerning ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-Seq approaches lack the ability to distinguish between fragments protected by ribosomes in active translation or by inactive ribosomes. To overcome these significant limitation, we developed RiboLace: a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied bothin vitroandin vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

Number and activity of active ribosomes in bacterial polyribosomes

Polyribosomes were isolated from Baccillus licheniformis, grown at 37 and 46 degrees C, and from Bacillus stearothermophilus, grown at 46 and 55 degrees C. The polyribosomes were incubated with either [3H]puromycin or [14C]phenylalanine. The number of active ribosomes (i.e. those to which growing polypeptide chains are attached) was calculated from the amount of [3H]peptidyl-puromycin formed. The activity of an active ribosome (i.e. the total number of amino acid molecules incorporated/unit time per active ribosome) was calculated from the uptake of [14C]phenylalanine. The number of active ribosomes per migrogram of RNA was as follows: for B. licheniformis, 1.66 × 10(12) and 1.72 × 10(12) at 37 and 46 degrees C respectively; for B. stearothermophilus, 2.59 × 10(12) at 46 and 55 degreesC respectively. The activity per active ribosome was as follows: for B. licheniformis, 0.61 and 0.05 at 37 and 46 degrees C respectively; for B. stearothermophilus, 0.58 and 0.42 at 46 and 55 degrees C respectively.