Mohr G, Rennard R, Cherniack AD, Stryker J, Lambowitz AM.
Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns. J Mol Biol. 307 (1) :75-92.
AbstractThe Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.
Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM.
Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol. 19 (12) :1162-7.
AbstractMobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.
Singh NN, Lambowitz AM.
Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference. J Mol Biol. 309 (2) :361-86.
AbstractGroup II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.
Caprara MG, Myers CA, Lambowitz AM.
Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions. J Mol Biol. 308 (2) :165-90.
AbstractThe Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron's catalytic core. Previous studies suggested a model in which the protein binds first to the intron's P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr).
Matsuura M, Noah JW, Lambowitz AM.
Mechanism of maturase-promoted group II intron splicing. EMBO J. 20 (24) :7259-70.
AbstractMobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.
Dickson L, Huang HR, Liu L, Matsuura M, Lambowitz AM, Perlman PS.
Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites. Proc Natl Acad Sci U S A. 98 (23) :13207-12.
AbstractGroup II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.