Role of helical constraints of the EBS1–IBS1 duplex of a group II intron on demarcation of the 5′ splice site

Abstract

Recognition of the 5′ splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem-loop of domain 1 and a complementary sequence at the 3 ′ end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem-loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5 ′ splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1-IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.

Structure of RNA Stem Loop B from the Picornavirus Replication Platform

“…Flexible regions within an RNA structure can be identified via analysis of chemical shift, resonance intensity, and residual dipolar couplings. Chemical shifts from highly flexible regions tend toward the chemical shifts of NMPs. , The differences between the carbon ( 13 C) chemical shifts of SLB and NMP values are shown in Figure A. As expected, the loop region displays chemical shifts more similar to those of the NMPs.…”

“…Chemical shifts from highly flexible regions tend toward the chemical shifts of NMPs. 29,30 The differences between the carbon ( 13 C) chemical shifts of SLB and NMP values 31 are shown in Figure 5A. As expected, the loop region displays chemical shifts more similar to those of the NMPs.…”

“…Flexible regions are also expected to produce narrow, intense resonances. ,,, Figure B displays intensities from a 1 H– 13 C HSQC spectrum. Resonance intensities for the loop region and terminal region are noticeably higher than those of the helix.…”

In vitroevolution of distinct self-cleaving ribozymes in diverse environments

“…A truncated ribozyme sequence containing only the proposed three-way junction retains catalytic activity (Supplementary Figure S7) supporting the functional significance of the three-way junction. The single stranded sequence within this three-way junction (3WJ-AGUAN) has sequence complementarity to the sequence within the conserved stem-loop, potentially forming a pseudoknot (Figure 4 ) in which the extent of pairing is demarcated by the helical constraints of pairing in the context of a stem loop ( 47 ). Several potential AC and GA pairs are present within this motif (Supplementary Figure S6).…”

NMR Studies of the Structure and Function of the HIV-1 5′-Leader

“…Although NMR spectroscopy has historically been used to determine structures of relatively small RNAs that typically comprise fewer than 60 nucleotides [67,68,69,70,71,72,73,74,75], the above studies have shown that structures can be probed by NMR spectroscopy in RNAs comprising up to 688 nucleotides. When used in combination with cryo-electron microscopy (cryoEM) or small-angle X-ray scattering (SAXS), 3D structural information can be obtained for relatively large protein:RNA complexes [68,69].…”