title: 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.
authors.affiliation: Institute for Cellular and Molecular Biology, Departments of Molecular Biosciences and Chemistry, University of Texas at Austin, Austin, TX 78712.
authors.forename: David M
authors.initials: DM
authors.lastname: Truong
authors.forename: David J
authors.initials: DJ
authors.lastname: Sidote
authors.forename: Rick
authors.initials: R
authors.lastname: Russell
authors.forename: Alan M
authors.initials: AM
authors.lastname: Lambowitz
mesh_terms: D001709:Biotechnology / Q000379:methods; D015152:Blotting, Northern; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics*; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D018626:Retroelements / Q000235:genetics*
chemical_list: D017931:DNA Primers; D016337:RNA, Catalytic; D018626:Retroelements; D008274:Magnesium
keywords: Mg2+ transport; RNA structure; directed evolution
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