FIGURE 5.

PABLO analysis reveals no evidence for 5′-pyrophosphate removal. (A) Schematic outline of the PABLO analysis for determination of the 5′-phosphorylation state of RNA. T4 DNA ligase joins oligo X₈₀ to 5′-mono-phosphorylated, but not to 5′-tri-phosphorylated RNA annealed to the bridging oligo Y. If ligation occurs an extended DNA–RNA chimera can be detected by either Northern blot analysis using a probe or directly on the autoradiogram, when oligonucleotide X₈₀ is radioactively labeled. (B,C) Tri-phosphorylated PPP-0536-RNA (lane 1) or different mixtures ([lanes 2,3] 65%; [lane 4] 50%; [lane 5] 33%; [lane 6] 20%; [lane 7] 10%; [lane 8] 5%; [lane 9] 2%; [lane 10] 1% mono-phosphorylated P-0536-RNA) of mono- and tri-phosphorylated 0536-RNA (lanes 2–10) were used to determine the efficiency of the PABLO assay. No Y-oligo (lane 2) or Y_{invitro_0536} (lanes 1,3–10) was added to the ligation mixture. Detection of the ligation products was achieved by Northern blot analysis (B) or directly on the autoradiogram as described above (C). (Arrows) The 0536-RNA, the radioactively labeled X₈₀ oligo, and the 0536-RNA-X₈₀ ligation product. (D,E) Partially mono-phosphorylated (lanes 1,2) and total RNA (RNA_T) from S. solfataricus (lanes 3–8) was subjected to PABLO analysis. No Y-oligo (lanes 1,3), Y_{invitro_0536} (lanes 2,4), Y_ATG (lane 5), Y_GATG (lane 6), Y_AGATG (lane 7), or Y_GAGATG (lane 8) was added to the ligation mixture. Detection of the ligation products was achieved by Northern blot analysis (D) or directly on the autoradiogram as described above (E). (Arrows) The 0536-RNA, the radioactively labeled X₈₀ oligo, and 0536-RNA-X₈₀ ligation product.