Figure 4.

(A) DNA template to generate RNAs containing both a ribozyme and an ORF encoding the polypeptide substrate of the ribozyme. Plasmid pTHF–GFPuv encodes the ribozyme and MKY peptide fused to six histidine residues, the FLAG epitope, and GFPuv (CLONTECH). The T7 polymerase promoter (T7) facilitated in vitro transcription and over expression in the appropriate bacterial strain. (B) Intracellular RNA-protein ligation. Histidine-tagged protein from E. coli cells overexpressing pTHF–GFPuv encoding both the ribozyme and THF–GFPuv substrate (+Ribozyme) or an analogous plasmid that had the ribozyme deleted (−Ribozyme) were affinity purified, and RNA fragments covalently attached to the protein were labeled at their 3′-end with cordycepin. Half of the cordycepin-labeled material isolated from cells overexpressing 21.4 and THF–GFP protein was treated with Proteinase K.