Atomic Force Microscopy Reveals DNA Bending during Group II Intron Ribonucleoprotein Particle Integration into Double-Stranded DNA

Abstract

The mobile Lactococcus lactis Ll.LtrB group II intron integrates into DNA target sites by a mechanism in which the intron RNA reverse splices into one DNA strand, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end to prime reverse transcription of the inserted intron RNA. These reactions are mediated by an RNP particle that contains the intron-encoded protein and the excised intron lariat RNA, with both the protein and base pairing of the intron RNA used to recognize DNA target sequences. Here, computational analysis indicates that Escherichia coli DNA target sequences that support Ll.LtrB integration have greater predicted bendability than do random Escherichia coli genomic sequences, and atomic force microscopy shows that target DNA is bent during the reaction with Ll.LtrB RNPs. Time-course and mutational analyses show that DNA bending occurs after reverse splicing and requires subsequent interactions between the intron-encoded protein and the 3'-exon, which lead to two progressively larger bend angles. Our results suggest a model in which RNPs bend the target DNA by maintaining initial contacts with the 5' exon, while engaging in subsequent 3'-exon interactions that successively position the scissile phosphate for bottom-strand cleavage at the DNA endonuclease active site and then reposition the 3' end of the cleaved bottom strand at the reverse transcriptase active site for initiation of cDNA synthesis. Our findings indicate that bendability of the DNA target site is a significant factor for Ll.LtrB RNP integration.Mobile group II introns, which are found in bacterial and organelle genomes, are retroelements that integrate site-specifically ("retrohome") into DNA target sites at high frequency and retrotranspose to ectopic sites that resemble the normal homing site at low frequency (reviewed in refs (1-4)). The integration reactions are mediated by an RNP complex that is formed during RNA splicing and contains the intron-encoded protein (IEP) 1 and the excised intron lariat RNA (3,4). RNPs initiate intron mobility by recognizing relatively long (30−35 bp) DNA target sites, with both the IEP and base pairing of the intron RNA contributing to the recognition of DNA † This work was supported by NIH grant GM37949 and Welch Foundation Grant F-1607 to A.M.L. and by support from the Welch NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript target sequences (5-8). Then, the intron RNA reverse splices into one strand of the DNA and is reverse transcribed by the IEP, yielding an intron cDNA that is integrated into the recipient genome by host DNA recombination or repair mechanisms (reviewed in refs 3,4). Their very high integration frequencies and target specificity combined with the ability to program insertion into different target sites by modifying the base-pairing sequences of the intron RNA have made it possible to develop mobile group II introns into highly efficient bacterial gene targeting vec...

Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

“…Additionally, decreased efficiency could result from suboptimal base-pairing interactions with the intron RNA, deleterious effects of nucleotide substitutions on intron RNA activity, occlusion of some target sites by proteins, or contributions of other factors, such as duplex stability or higher-order DNA structure (30). Recent studies showed that integration of Ll.LtrB intron RNPs results in bending of the target DNA, and computational analysis suggested that DNA bendability in addition to sequence may influence DNA integration efficiency (29). We anticipate that the algorithm will continue to improve as larger databases become available and more information about other factors is incorporated.…”

Group II Introns: Mobile Ribozymes that Invade DNA

“…Bottom-strand cleavage occurs at position +9 from the insertion site and requires additional interactions between the IEP and the 3 ′ exon, the most critical being recognition of T+5 (Singh and Lambowitz 2001). Binding of the RNP to the 5 ′ and 3 ′ DNA exons bends the target DNA, with the bend angle increasing when the cleaved 3 ′ end is repositioned from the En to RT active site for initiation of reverse transcription (Noah et al 2006). Because reverse splicing into the DNA target site is reversible and the equilibrium favors intron excision, the reaction must be driven forward by the initiation of cDNA synthesis, which blocks the 3 ′ -splice site and prevents excision (Aizawa et al 2003).…”

Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution

“…DIVa nevertheless remains a high-affinity binding site for the IEP, reflecting a critical role of this interaction in nucleating RNP assembly and positioning the IEP for initiation of TPRT (65). After splicing, the IEP remains tightly bound to the intron lariat in a stable RNP that promotes retrohoming (30,42).…”

“…Bottom-strand cleavage between positions +9 and +10 of the DNA target site occurs after a lag and requires additional interactions of the IEP with the 3′ exon, most critically recognition of T+5. Atomic force microscopy showed that the binding of RNPs bends the target DNA into two progressively sharper bend angles, the first correlated with 3′-exon interactions that position the scissile phosphate at the En active site for bottom-strand cleavage, and the second with repositioning of the 3′ end of the cleaved strand from the En to the RT active site for initiation of cDNA synthesis (42). Notably, even with this DNA bending, the distance between DNA target site residues T-23 and T+5 recognized by the IEP is longer than can be spanned readily by a single IEP molecule.…”

“…The IEP also makes weaker secondary contacts with conserved core regions, including in DI, DII, and DVI, that stabilize the active ribozyme structure for RNA splicing and reverse splicing (31,40). This mode of interaction in which the IEP is anchored by binding to DIVa and binds more weakly to the catalytic core may enable the IEP to accommodate conformational changes within the intron RNA during RNA splicing and different steps in intron mobility (31,36,41,42,43).…”