High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA.
Mobile group II introns encode proteins that have reverse transcriptase and maturase activities and bind specifically to the intron RNA to promote both RNA splicing and intron mobility. Previous studies with the Lactococcus lactis Ll.LtrB intron showed that the intron-encoded protein (LtrA) has a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure containing the translation initiation region of the LtrA open reading frame, and that this binding site consists of a small stem-loop emanating from a purine-rich internal loop. The binding of LtrA to DIVa is important for translational regulation, RNA splicing, and intron mobility. Here, we show by in vitro selection that part of the purine-rich internal loop can be closed by base pairing, enabling the LtrA binding site to be represented as an extended stem-loop structure with a bulged A (A556) required for tight binding of LtrA. The deletion or pairing of A556 has relatively little effect on maturase-promoted RNA splicing, but significantly inhibits intron mobility. The wild-type DIVa structure has a second bulged A (A553), which is selected against in tightly binding variants. As expected from the selection, the deletion or pairing of A553 results in tighter binding of LtrA, but surprisingly, also inhibits intron mobility. These findings suggest that the binding of LtrA to DIVa is delicately balanced, so that either too weak or too tight binding can be deleterious. The nature of the maturase/DIVa interaction and its role in translational regulation are reminiscent of the coat protein/RNA hairpin interactions of single-stranded RNA phages.
Watanabe
Kazuo
K
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, School of Biological Sciences, University of Texas at Austin, 78712, USA.
Lambowitz
Alan M
AM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D012329:RNA, Bacterial; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase