Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

Abstract

Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg 2+ concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg 2+ transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg 2+ concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg 2+ concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg 2+ binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg 2+ -dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg 2+ concentrations, with implications for evolution and potential applications in gene targeting. directed evolution | Mg 2+ transport | RNA structure M obile group II introns are retrotransposons that are found in prokaryotes and the mitochondrial and chloroplast DNAs of some eukaryotes and are thought to be evolutionary ancestors of spliceosomal introns, the spliceosome, retrotransposons, and retroviruses in higher organisms (1). They consist of two components-an autocatalytic intron RNA ("ribozyme") and an intron-encoded protein (IEP) with reverse transcriptase activity-that function together in a ribonucleoprotein (RNP) complex to promote RNA splicing and site-specific integration of the intron into new DNA sites in a process called retrohoming (1). Like spliceosomal introns, group II introns splice via two sequential transesterification reactions that yield an excised intron lariat RNA (2). For group II introns, the splicing reactions are catalyzed by the intron RNA with the assistance of the IEP, which binds specifically to the intron RNA and stabilizes the catalytically active RNA structure. The IEP then remains bound to the excised intron lariat RNA in an RNP that promotes retrohoming via reverse splicing of the intron RNA directly into DNA sites followed by reverse transcription by the IEP. The resulting intron cDNA is integrated into the genome by host enzymes (3, 4). The ribozyme-based, site-specific DNA integration mechanism used by g...

Genome-Wide Analysis of RNA Secondary Structure

“…PARS has also been adapted to include PARTE (parallel analysis of RNA structure with temperature elevation), which allows calculation of RNA folding energies by measuring RNA structures at different temperatures (119). These three methods have been applied using a biological pH (pH 7-7.5) but at typical in vitro structure-probing concentrations of Mg 2+ (5-10 mM) and monovalent salt (100-150 mM NaCl), which are well above the physiological levels of Mg 2+ of ∼0.5-1 mM in eukaryotes (65) and 2 mM in prokaryotes (71,111). Although many interesting findings arise from observations of RNA structure, one key challenge is to understand the role proteins play in affecting RNA structure.…”

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

“…We chose to evaluate RNA folding in 0.5 and 2 mM Mg 2+ , as these are within the range of free Mg 2+ concentrations reported in eukaryotic and prokaryotic cells, respectively, and in 150 mM K + , as this is near physiological (Lusk et al 1968;London 1991;Alberts et al 1994;Feig and Uhlenbeck 1999;Grubbs 2002;Romani 2007;Truong et al 2013). Additionally, we examined folding in 140 mM KCl without Mg 2+ added in order to later determine if molecular crowders and cosolutes could promote folding cooperativity in the absence of divalent ions.…”

“…Physiological conditions are quite different. Typical K + concentrations in prokaryotic and eukaryotic cells are only ∼140 mM, while free Mg 2+ concentrations are just 1.5-3.0 mM in prokaryotic cells (Lusk et al 1968;Truong et al 2013) and 0.5-1.0 mM in eukaryotic cells (London 1991;Alberts et al 1994;Feig and Uhlenbeck 1999;Grubbs 2002;Romani 2007). Eukaryotic and prokaryotic cells also contain 20%-40% (w/v) macromolecules, as well as lower-3 molecular-weight cosolutes, which can exclude volume, interact with the RNA, and lead to altered solvent properties (Ellis 2001;Minton 2001).…”

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes

“…It has been shown both in bacterial and eukaryotic cells that the efficiency of retrohoming is strongly coupled to the Mg 2ϩ concentration in the cell (47,48). In fact, the lower Mg 2ϩ concentration of the eukaryotic cell limits the retrohoming efficiency of group II introns that are of bacterial origin.…”

“…Moreover, several metal ion binding sites have been located in the active site (38 -40), and a two-metal ion mechanism (41,42) has been suggested to underlie intron catalysis (43,44). In-cell studies establishing a correlation between the intracellular Mg 2ϩ concentration and the frequency of splicing and retrohoming buttress the relevance of Mg 2ϩ for group II intron catalysis (45)(46)(47)(48). The importance of the identity of the divalent metal ions bound to the intron is underscored by the finding that the presence of Mn 2ϩ can lead to a shift of the cleavage site (49) and that already low amounts of Ca 2ϩ decrease the turnover rate by 50% in the Sc.ai5␥ intron (50).…”