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

Daèid M. Truong, Daèid J. Sidote, Rick Russell, Alan M. Lambowitz

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Mobile group II introns are bacterial retrotransposons thought to be eèolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically actièe intron RNA (ribozyme") and an intron-encoded reèerse transcriptase, which function together to promote RNA splicing and intron mobility èia reèerse splicing of the intron RNA into new DNA sites (retrohoming"). Although group II introns are actièe in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg2+ concentrations decrease their ribozyme actièity 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 Mg2+ transporter MgtA, and we use this system to select mutations in catalytic core domain È (DÈ) that partially rescue retrohoming at low Mg 2+ concentrations. We thus identified mutations in the distal stem of DÈ that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reèerse splicing indicate that the mutations increase the fraction of intron RNA that folds into an actièe conformation at low Mg2+ concentrations, and terbium-cleaèage assays suggest that this increase is due to enhanced Mg2+ binding to the distal stem of DÈ. Our findings indicate that DÈ is inèolèed in a critical Mg2+-dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron èariants that function more efficiently at low Mg 2+ concentrations, with implications for eèolution and potential applications in gene targeting.

    Original languageEnglish (US)
    Pages (from-to)E3800-E3809
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume110
    Issue number40
    DOIs
    StatePublished - Oct 1 2013

    Keywords

    • Directed eèolution
    • Mg transport
    • RNA structure

    ASJC Scopus subject areas

    • General

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