The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.
Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of =0.12 pM at 30 degrees C. This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.
M.Matsuura and J.W.Noah contributed equally to this workMobile group II introns encode reverse transcriptases that also function as intron-speci®c splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high af®nity 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 modi®cation 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.
Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.
Retrohoming of group II introns occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Host repair enzymes are predicted to complete this process. Here, we screened a battery of Escherichia coli mutants defective in host functions that are potentially involved in retrohoming of the Lactococcus lactis Ll.LtrB intron. We found strong (greater than threefold) effects for several enzymes, including nucleases directed against RNA and DNA, replicative and repair polymerases, and DNA ligase. A model including the presumptive roles of these enzymes in resection of DNA, degradation of the intron RNA template, traversion of RNA-DNA junctions, and second-strand DNA synthesis is described. The completion of retrohoming is viewed as a DNA repair process, with features that may be shared by other non-LTR retroelements. Movement of group II introns to allelic sites on DNA occurs via an RNA intermediate in a process termed retrohoming (for review, see Belfort et al. 2002;Lambowitz and Zimmerly 2004). Retrohoming pathways have been studied in detail for two related yeast mitochondrial introns (aI1 and aI2) and two bacterial introns (Ll.LtrB and RmInt1) (Belfort et al. 2002;Lambowitz and Zimmerly 2004; Martinez-Abarca et al. 2004). The Ll.LtrB intron, which derives from Lactococcus lactis (Mills et al. 1996;Shearman et al. 1996), was shown to be functional in both splicing and retrohoming in Escherichia coli (Mills et al. 1997;Cousineau et al. 1998). In all organisms, the retrohoming of group II introns occurs by a process in which the excised intron RNA reverse splices into one strand of a DNA target site and is then reverse transcribed by the intron-encoded protein (IEP). This process is mediated by a ribonucleoprotein (RNP) particle that is formed during RNA splicing and contains the IEP and the excised intron lariat RNA. In addition to catalytically active intron RNA, retrohoming of the Ll.LtrB intron is dependent upon three activities of the IEP (Belfort et al. 2002;Lambowitz and Zimmerly 2004;Lambowitz et al. 2005). These are RNA maturase, to stabilize the catalytically active structure of the intron RNA for RNA splicing and reverse splicing, DNA endonuclease, for cleavage of the target DNA to generate a primer for reverse transcription, and reverse transcriptase (RT) for making a cDNA copy of the intron RNA (Fig. 1).Completion of the retrohoming pathway in bacteria is distinguished from the major pathway in yeast by its independence of homologous recombination between donor and recipient (Eskes et al. 1997;Mills et al. 1997;Cousineau et al. 1998;. After complete reverse splicing of the Ll.LtrB intron and endonucleolytic cleavage of the second strand, 9 nucleotides (nt) downstream of the intron-insertion site, fulllength cDNA synthesis ensues in a process termed target DNA-primed reverse transcription (TPRT) (Fig. 1, steps 1-3). The later stages of retrohoming require degradation or displacement of...
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