Supplementary Data for: Crystal Structure of a Self-Splicing Group I Intron with Both Exons Peter L. Adams, Mary R. Stahley, Anne B. Kosek, Jimin Wang and Scott A. Strobel The supplementary material includes the following information: (i) Demonstration that the pre-2s complex forms properly in vitro; (ii) Exon ligation assays of the all ribose pre-2s complex and its 2'-deoxy ribose containing derivatives; (iii) Anomalous electron density maps of the heavy atom derivatives used to confirm the proper nucleotide register within the structure; (iv) Comparison of the Tl + anomalous electron density maps for the entire pre-2s complex with ribose or 2'-deoxy ribose at WG206; (v) A table of ligands and bond distances for metals M 1 and M 2 in the pre-2s complex with a 2'-deoxy ribose at WG206 (Table I). 1. Formation of the pre-2s RNA complex with U1A Formation of the pre-2s ternary complex between the chimeric oligonucleotide, the intron RNA transcript (UP62) and the U1A protein was confirmed by gel shift analysis (SupFig 1). Radiolabeled dcirc oligonucleotide (2.5mM) and UP62 RNA (5mM) were heated together at 50 C for 2 min and slow cooled. U1A protein (10mM) was added at room temperature. All 1
samples contained 50mM HEPES ph 6.5, 10mM MgCl 2, 25mM NaCl, 5% glycerol, and 0.02% xylene cyanol. RNA complexes were analyzed by 6% native PAGE (1x TBE, 10mM MgCl 2 ). Radiolabeled dcirc oligonucleotide alone migrates with mobility A (lane 1). Upon inclusion of a two-fold excess of UP62, the mobility of dcirc was quantitatively shifted to B (lane 2). U1A addition reduced the mobility still further (C, lane 3). This demonstrated formation of a homogenous complex between three of the crystallization components. In crystallographic trials, a slight excess of dcirc was used relative to UP62. Gel shift analysis was also used to titrate the U1A to the level sufficient for complete complex formation (data not shown). Efforts to monitor CAT binding by gel shift analysis were unsuccessful. 2. Activity of the pre-2s complex The activity of the pre-2s intron complex was tested using ribose and 2'-deoxy ribose substrates, in the absence and presence of the U1A protein (SupFig. 2). Reactions were performed in 50 mm sodium cacodylate ph 6.8, 10 mm MgCl 2, 1 µm UP62 RNA, 2µM CAU (r) or CAT (d) oligonucleotide, and 1.2 µm U1A protein. A trace amount of 5'-end 32 P- radiolabeled dcirc or rcirc was added to initiate the reaction. Using the ribose substrates (CAU and rcirc), the intron reached equilibrium between the forward and reverse reactions in less than 2 min (lane 2). This end point corresponded to 40% exon ligation, which is consistent with an internal equilibrium between the forward and reverse reaction of approximately 1 1. Neither mutation of the P6 loop to create a U1A binding site, nor addition of the U1A protein affected the rate at which this equilibrium was reached (lanes 2 and 3). 2'-Deoxy substitution at U-1in the 5'-exon slowed the rate 3000-fold, but the complex reached the same end point (lane 4). WdG206 substitution reduced the rate at least 10 6 fold and the equilibrium end point was 2
never established (lanes 5 and 6). This demonstrates that the all ribose and single U-1 2'-deoxy substituted complexes are reactive, while WdG206 substitution is sufficient to inactivate the intron. 3. Confirmation of RNA register The RNA register in the refined structure was confirmed by introducing two covalent substitutions within the RNA that have an anomalous signal: a 5-bromo substitution at dt-1 in the 5'-exon, and a 2'-selenomethyl substitution at C194 within the intron 2. Crystals were grown using either of these oligonucleotides in place of the native variants. Both derivatives produced crystals that were isomorphous with the native complex. The anomalous x-ray scattering electron density peaks correlated precisely with the expected location of these functional groups within the model (SupFig. 3a and b). The Br substitution at the 5'-splice site unequivocally established the location of the active site. The Se substitution established the register of dcirc, which by direct extension identified the boundary between the intron and the 3'-exon. 4. Tl + anomalous electron density for complexes with ribose or 2'-deoxy ribose at WG206 rcirc or dcirc containing pre-2s crystals were soaked with 2mM Tl +, and the sites of Tl + binding were identified by calculating an anomalous electron density map. This density was superimposed on the pre-2s structure (SupFig. 4). Tl + binding outside the active site was equivalent between the two crystal forms, but the strong Tl + site at active site position M 2 observed in the dcirc complex (SupFig. 4a) was completely eliminated in the rcirc complex (SupFig. 4b). This suggests that 2'-deoxy ribose substitution at WG206 results in monovalent metal ion binding to the active site. 3
Supplementary Figure Legends Supplementary Figure 1. Native gel shift analysis to demonstrate the assembly of UP62, dcirc and U1A into the pre-2s complex. The presence or absence of each component is indicated in the labeling above the figure. The dcirc oligonucleotide is radiolabeled in this experiment (indicated with the asterisk). The mobility labels used in the text are marked on the right of the autoradiogram. Supplementary Figure 2. Reactivity of the pre-2s complex containing ribose or 2'-deoxy at the ligation junction. The length of incubation time for each reaction is indicated, as well as the presence (+) or absence (-) of various components in the complex. In this experiment the 3'-exon oligonucleotide CIRC (d or r) oligonucleotide is 5'-end labeled. Reaction with the 5'-exon oligonucleotide (CAT or CAU) transfers the last six nucleotides onto the 5'-exon, which reduces the length of CIRC from 22 to 16 nucleotides. Supplementary Figure 3. Confirmation of RNA register by specific covalent attachment of atoms that anomalously scatter X-rays. a. 5-Bromo-2'-deoxy uridine substitution at dt-1 in oligonucleotide CAT. The bromine anomalous density peak (blue) is overlaid on the G10 dt-1 wobble pair (orange and red) at the 5' splice site. The density superimposes on the 5-methyl group of dt-1. b. 2'-Selenomethyl 2'-deoxy cytidine substitution at C194 in oligonucleotide dcirc. The selenium anomalous density (green) is overlaid on residues G193 through C195 (blue). The density superimposes on the 2'-OH of C194. 4
Supplementary Figure 4. Tl + anomalous electron density maps for dcirc (a) and rcirc (b) containing crystals. The overall structure of the intron (blue) and exons (red) are shown as a ribbon traced through the phosphate backbone. The density for Tl + is shown in orange contoured at 3.5s and 3.4s for dcirc and rcirc, respectively. Supplementary Material References 1. Mei, R. & Herschlag, D. Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron. Insights into catalysis and the second step of self-splicing. Biochemistry 35, 5796-5809 (1996). 2. Höbartner, C. & Micura, R. Chemical Synthesis of Selenium-Modified Oligoribonucleotides and Their Enzymatic Ligation Leading to an U6 SnRNA Stem- Loop Segment. J Am Chem Soc 126, 1141-1149 (2004). 3. Harding, M. M. The geometry of metal-ligand interactions relevant to proteins. Acta Crystallogr D Biol Crystallogr 55 ( Pt 8), 1432-43 (1999). 4. Harding, M. M. Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr D Biol Crystallogr 58, 872-4 (2002). 5
Table 1. Potential ligands and bond distances for active site metal ions M 1 and M 2 in the pre-2s complex with an WdG206 substitution Potential Ligands Distance (Å) 1 C88 O2P J5/4 2.4 G170 O2P J8/7 2.7 A172 O2P J8/7 2.1 U-1 O3' 5'-exon 2.4 A+1 O1P 3'-exon 2.0 U173 O2P J8/7 (outer sphere) 4.4 Standard Mg 2+ ligand distances 3 2.0 2.4 2 A87 O2P J5/4 3.0 A128 O2P J6/7 2.4 A127 O2' J6/7 2.5 A172 O1P J8/7 3.0 W 1 2.9 W 2 3.0 WG O2' (predicted) 2.8 Standard K + ligand distances 4 2.4 3.2 6
U1A UP62 dcirc* 1 2 3 C B A P. Adams, et al. (2004) Supplementary Figure 1
Time (min) UP62 U1A rcirc or dcirc CAU or CAT 0 3 3 3000 3000 3000 0 - + + + + + - - - + + + + - r r r r d d d - U U T U T - 1 2 3 4 5 6 7 Substrate (22nt) - Product (16nt) - P. Adams, et al. (2004) Supplementary Figure 2
a b P. Adams, et al. (2004) Supplementary Figure 3
P. Adams, et al. (2004) Supplementary Figure 4