Effects of Maturase Binding and Mg²⁺ Concentration on Group II Intron RNA Folding Investigated by UV Cross-Linking

Abstract

The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure. Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein. Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure. In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein. The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing. These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity. Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible. This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing. The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture. Finally, an abundant central wheel cross-link, present in >50% of the molecules at 5 mM Mg(2+), suggests models in which group II intron domains I and II are either coaxially stacked or aligned in parallel, bringing the 5'-splice site together with the 3'-splice site and catalytic core elements at JII/III. This and other cross-links provide new constraints for three-dimensional structural modeling of the group II intron catalytic core.

A DEAD-box protein alone promotes group II intron splicing and reverse splicing by acting as an RNA chaperone

“…These differences suggest that the active structures of aI5␥ and bI1 at physiological KCl and Mg 2ϩ concentrations may differ in significant ways from those formed under self-splicing conditions. A similar conclusion was reached for the maturase-promoted and selfsplicing reactions of the Ll.LtrB intron based on chemical structure mapping and analysis of UV-induced RNA-RNA cross-links (7,8). We suggested previously that inactive structures stabilized by high Mg 2ϩ concentrations might contribute to the strongly decreased rate of self-splicing compared with maturase-dependent splicing of that intron (7,8).…”

“…Until now, a protein-dependent in vitro splicing system has been developed only for the RT͞maturase protein encoded by the mobile Lactococcus lactis Ll.LtrB intron (5, 6). Studies using this system showed that the maturase binds specifically to the intron RNA and promotes its splicing by stabilizing the catalytically active RNA structure (7,8).In addition to proteins that stabilize specific RNA structures, cellular RNAs may require RNA chaperones to disrupt stable inactive structure (''kinetic traps'') during RNA folding (9, 10). In the case of group I and II introns, this RNA chaperone function is thought to be fulfilled by specific DExH͞D-box proteins (11, 12).…”

“…Until now, a protein-dependent in vitro splicing system has been developed only for the RT͞maturase protein encoded by the mobile Lactococcus lactis Ll.LtrB intron (5, 6). Studies using this system showed that the maturase binds specifically to the intron RNA and promotes its splicing by stabilizing the catalytically active RNA structure (7,8).…”

The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function

“…The splicing of the group II intron aI2 depends on the intron-encoded p62 maturase, which by analogy to the Lactobacillus lactis Ll.LtrBencoded maturase (23,24) likely functions by stabilizing the catalytically active structure of the intron RNA. We were intrigued by the finding that expression of one copy of CYT-19 in mss116⌬ grown at 30°C restores wild-type levels of p62 (Fig.…”

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

“…Protein concentrations were determined by Bradford assay (21), using LtrA protein whose concentration had been determined by using A 280 as a standard. The protein preparations were >98% pure, as judged by Coomassie blue-stained SDS-polyacrylamide gels Ll.LtrB lariat RNA was generated by self-splicing of a 971-nt Ll.LtrB RNA (902-nt Ll.LtrB intron flanked by 5'-and 3'-exon sequences of 32 and 37 nt, respectively) transcribed from BamHI-linearized pJNΔORF with phage T7 RNA polymerase, using a MEGAScript kit (Ambion, Austin, TX) (22). Self-splicing was carried out with 2 μM Ll.LtrB RNA in 1 ml of 0.5 M NH 4 Cl, 50 mM MgCl 2 , 40 mM Tris-HCl, pH 7.5 at 30°C.…”