Supplementary Figure 1: RNP purification, components and activity.
From: Structure of a group II intron in complex with its reverse transcriptase
(a) Intein-mediated RNP production and purification. The Ll.LtrB intron RNA (red) (Exons E1 and E2 in green) and associated intron encoded protein (LtrA in yellow) were co-expressed with nisin induction. The RNP was captured and purified on chitin resin column through the chitin binding domain (CBD) that is fused to the LtrA C-terminus, released by DTT cleavage of the intein (I), and further purified by sucrose gradient sedimentation. (b) Product purity. LtrB intron RNA (902 nt) and associated LtrA protein (~70 kD) were analyzed, respectively, on a 1.2% denaturing formaldehyde agarose gel (left, lane 1, R) and on 12% denaturing SDS-PAGE gels (right, lanes 1, P), stained with Coomassie (blue) and silver (brown). Silver staining also revealed a higher molecular weight product, likely corresponding to intron RNA which stains with silver, not seen in a purified LtrA control (lanes 2). (c) RNA analysis. RNA in purified spliced RNPs (lanes 3 and 5) or extracted from the RNP (lane 4) were assayed by primer extension and the cDNA analyzed on an 8% urea- PAGE gel. RNPs were either wild-type (wt) or a splicing-defective mutant of the catalytic triad (Triad). A primer, specific to the 5' end of the intron (black arrow and P), was used to reveal precursor (pre-mRNA) and spliced intron, from which 68-nt and 41-nt cDNA products, respectively, were generated. Lane 1 corresponds to total RNA isolated from cells expressing the group II intron RNP and lane 2 to no RNA. The purified RNP resulted in predominantly a 41-nt band with a minor amount of precursor (lane 3), indicating that most of the pre-mRNA had been removed. In contrast, the splicing defective Triad RNP yielded a primer extension product that corresponded exclusively to the intron-containing precursor (lane 5). (d) Activity assays with target DNA. Purified RNPs incubated with target DNA (T, 52nt) were assayed for DNA binding (left) and reverse splicing activity (right) on a 4% native-PAGE and a 10% urea-PAGE gel, respectively. Lanes 1-3 contain no RNP, wild-type RNP and RNP from the catalytic triad mutant, respectively. Only the wt RNP, and not the catalytic mutant Triad, yielded shifted target DNA (left, black triangles). Likewise, only the wt RNP was active in reverse splicing (right) on the target DNA (IS=integration site, CS=cleavage site), producing a high molecular-weight integration product (black triangle) and the corresponding reverse splicing product (RSP) and cleavage product (CP).
