Structural accommodations accompanying splicing of a group II intron RNP

Figure 1.. Activity and composition of a group II intron RNP. (A) Group II intron's cycle of activities. The intron RNA is shown in gray. Dark pink and gold denote exons 1 and 2, respectively. The branch point adenosine is shown as a circled ‘A’. The transcribed precursor folds with help of the IEP (green) and undergoes two consecutive transesterification reactions to release the intron lariat from the exons that become ligated. The intron is then capable of recognizing and integrating into target DNA (blue) by first reverse-splicing into the top strand, followed by bottom strand cleavage and target-primed reverse transcription (TPRT, product shown in black). (B) Secondary structure of group II intron Ll.LtrB. DI of the intron RNA is shown in light gray and the rest of the domains are shown in dark gray. The tertiary interactions are indicated by dotted lines. The IEP anchor site and the action interface are boxed. DIVb is omitted for simplicity. (C) EBS-IBS interactions between the Ll.LtrB intron RNA and the exons. Blue dashed line denotes exon ligation site. The black dot denotes the G-U pair between EBS1 and IBS1. (D) Domain organization of the Ll.LtrB intron IEP (LtrA). NTE: N-Terminal…

Figure 2.. Group II intron Ll.LtrB RNP in pre- and post-catalytic states. (A) Spliced intron RNP (+A, right) and the two precursor RNPs. ΔA and Triad mutants are trapped in the pre-catalytic state by deleting the bulged branch-point adenosine in DVI or mutating the catalytic triad sequences in DV (AGC>AAU), respectively. (B) Splicing analysis by primer extension of total RNA isolated from the three constructs. The +A construct is capable of splicing whereas ΔA and Triad mutants generate little (ΔA) or no observable (Triad) excised intron. (C) Native gel showing substrate DNA binding to the purified RNPs. The +A RNP is able to bind the target DNA, whereas ΔA and Triad RNPs are not capable of DNA binding. (D) Size-Exclusion Chromatography (SEC) characterization of the Triad and +A RNPs. SEC was carried out at 25°C. The precursor RNP was incubated on ice or at 37°C for 15 min (with or without another 15 min on ice) or at 30°C for 3 h. Since equilibrium measurements were not done, the SEC data do not reflect the actual ratio of dimers and monomers in a given condition. The color code for the treatments is shown on the right. Retention times for globular protein standards are indicated with…

Figure 3.. DNA oligonucleotide binding shows increased nucleotide accessibility in DId of the post-catalytic RNP. (A) Schematic showing RNA regions to which the DNA oligonucleotide probes are complementary. The DNA oligonucleotides were designed to cover the entire RNA from 5′ to 3′. Briefly, the 32P-labeled oligonucleotides were co-incubated with the RNP at 37°C for 1 h and separated on a native 4% polyacrylamide gel for binding comparison. (B) Quantitative analysis of DNA oligonucleotide binding to Triad and +A RNPs. The binding experiment, exhibiting different levels of binding affinity in DI, was conducted three times and the data were averaged. Representative gels are shown for DNA oligonucleotides covering key regions of the RNA. Results of the ΔA RNP are similar to the Triad RNP and the binding patterns for all three RNPs are shown in Supplementary Figure S3C. The results using loop-specific tiling probes and data for experiments carried out at different temperatures are shown in Supplementary Figure S3D and E, respectively. (C) Spatial locations of the nucleotide accessibility gain in DId as reflected by DNA oligonucleotide probing. RNA nucleotides with moderately (pink) and…

Figure 4.. SHAPE profile of the +A RNP and comparison to the Triad RNP. (A) Representative gels showing in vitro SHAPE reactivity of the EBSs and DI(iii)a nucleotides in the +A RNP and the Triad RNP, as well as data for the +A RNP in vivo. Structures and colored dots are as in panel B. (B) Secondary structure of the Ll.LtrB intron showing in vitro SHAPE profile of the +A RNP and the comparison to that of the Triad RNP. Colored dots are assigned to nucleotides to denote the levels of SHAPE modifications. SHAPE reactivity comparison is shown by coloring nucleotides with either orange or cyan in the +A profile to represent increase or decrease compared to that of the Triad RNP, respectively. DVI nucleotides in light gray denote the site where the +A RNP was not profiled. The truncated DIVb is mostly omitted. Representative data for all three RNPs are shown in Supplementary Figure S4. Shaded nucleotides are postulated protein-interaction sites within an arbitrary 4-Å distance from the protein in the cryo-EM structure (9) (PDB: 5g2x).

Figure 5.. SHAPE profile of +A RNP is largely in accord with cryo-EM structure (9) (PDB: 5g2x). Bases are shown for nucleotides involved in key tertiary pairings and/or those with SHAPE reactivity (colored in red). (A) Tertiary base pairing β–β′ (blue) is protected from SHAPE chemistry while adjacent nucleotides are modified. (B) SHAPE modifications near the two tetraloop-receptor interactions between DII and DVI. The π–π′ interaction nucleotides (upper pair) show no SHAPE reactivity, whereas nucleotides involved in the η–η′ interaction (lower pair) show sensitivity to SHAPE. (C) IEP anchor site in DIVa is largely protected by protein interaction. The Shine-Dalgarno (SD) sequence is shown as a bold tube. The AAAA loop is indicated by black arrows. (D) DI(iii)a is sensitive to SHAPE despite being vicinal to the Fingers/Palm domain of the IEP. Insertion 4a is shown in blue with its unresolved part denoted as a dotted line.

Figure 6.. Molecular dynamic simulations of the +A RNP. (A) Domain fluctuations of the intron RNA highlighting nucleotides with moderate (green), high (orange) and very high (red) fluctuations. Nucleotides with RMSF below 0.3 are shown in dark blue. Tertiary interaction motifs and the domains are noted. (B) Fluctuation correlations among domains in the intron RNA, the exons and the IEP. Pale-red (1.0) and blue-green (–0.5) colors indicate high and low ends of correlation values, respectively. Fingers/Palm domain is divided at the insertion 4a truncation (Y252-L301 as shown in Supplementary Figure S8A), with Fingers/Palm 1 adjacent to the NTE and Fingers/Palm 2 close to the Thumb domain at the action interface. The black and blue arrows indicate representative tertiary and protein interactions, respectively.

Figure 7.. Putative structural rearrangements in transition between the two catalytic states. (A) Key regions with gained SHAPE reactivity in the +A RNP are located at the action interface of the RNP. Retained mRNA is shown in magenta. Light and dark green represent the NTE and the Fingers/Palm domain, respectively. Blue denotes the Thumb domain, cyan denotes the DNA binding domain and orange denotes the DNA endonuclease domain. The boxes indicate the action interface (merlot) and the IEP anchor (green). (B) Spatial arrangements of κ–(ii)a–ζ region and the adjacent DVI at the active site. In the post-catalytic RNP the κ loop has strengthened tertiary interactions with κ′, indicated by red arrows. ζ–ζ′ also has strengthened interactions as do A205-A206 and U195-U194 (green). DV is shown in black. Light blue, cyan and dark pink represent the κ–κ′, ζ–ζ′ and μ–μ′ interactions, respectively. The catalytic triad nucleotides are shown as a bold tube. 5′ and 3′ ends are shown in red and J2/3 is shown in orange.