Figure 2.

Alternative tags for purifying ribosomes. The standard TRAP protocol (gray box) can be adapted for use with a variety of ribosomal tags. A, The RPL10A/GFP construct tags the large (60S) subunit, which does not engage mRNA until initiation of translation. Thus, it will not capture scanning small subunit (40S) particles before initiation, although it will capture 60S particles unaffiliated with mRNA. B, The HA-tagged L22 protein from the Ribotag mouse also tags the large subunit and should have similar consequences. C, The S6 protein of the small subunit is phosphorylated in response to activity. Thus, capture of phospho-S6 should yield translating as well as potentially scanning or stalled mRNA from activated cells. It is speculated that mRNA capture from each cell will be proportional to the amount of S6 phosphorylation, and thus activity. D, Tagging of RPL10A with a nanobody (NB) against GFP allows the conversion of any GFP expressing virus or mouse line into a TRAP reagent. In this system, soluble GFP in the cell provides a linker for the tagged ribosome and the anti-GFP bead. However, as GFP is soluble ex vivo, careful consideration must be given to blocking excess GFP.