Statistical modeling and analysis of the LAGLIDADG family of site- specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family

The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis , regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition–fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes.

Section: Resultsmentioning

confidence: 99%

Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria

Salman

Amann

Shub³

et al. 2012

“…The Ho protein has the same two domains but lacks a host gene and has an additional zinc finger domain at its C terminus (23). Within the phylogenetic tree of all LAGLIDADG endonucleases (26), Ho clusters with the VMA1 intein (called VDE or PI-SceI), which is the only true intein in the S. cerevisiae genome. The VMA1 intein, like HO, is found only in hemiascomycetes, although its taxonomic distribution has been affected by horizontal transmission between species (27,28).…”

Section: Acquisition Of the Ho Endonuclease Gene In Species Close To Smentioning

confidence: 99%

Evolution of the MAT locus and its Ho endonuclease in yeast species

Butler

Kenny

Fagan

et al. 2004

220

225

The genetics of the mating-type (MAT) locus have been studied extensively in Saccharomyces cerevisiae, but relatively little is known about how this complex system evolved. We compared the organization of MAT and mating-type-like (MTL) loci in nine species spanning the hemiascomycete phylogenetic tree. We inferred that the system evolved in a two-step process in which silent HMR͞HML cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element. Ho-mediated switching between an active MAT locus and silent cassettes exists only in the Saccharomyces sensu stricto group and their closest relatives: Candida glabrata, Kluyveromyces delphensis, and Saccharomyces castellii. We identified C. glabrata MTL1 as the ortholog of the MAT locus of K. delphensis and show that switching between C. glabrata MTL1a and MTL1␣ genotypes occurs in vivo. The more distantly related species Kluyveromyces lactis has silent cassettes but switches mating type without the aid of Ho endonuclease. Very distantly related species such as Candida albicans and Yarrowia lipolytica do not have silent cassettes. In Pichia angusta, a homothallic species, we found MAT␣2, MAT␣1, and MATa1 genes adjacent to each other on the same chromosome. Although some continuity in the chromosomal location of the MAT locus can be traced throughout hemiascomycete evolution and even to Neurospora, the gene content of the locus has changed with the loss of an HMG domain gene (MATa2) from the MATa idiomorph shortly after HO was recruited.M ating in Saccharomyces cerevisiae is a choreographed fusion of two haploid cells with opposite mating types to form a diploid zygote. The mating type of a haploid cell is determined by its genotype at the mating-type (MAT) locus on chromosome III. The two variants of the MAT locus, MAT␣ and MATa, are referred to as idiomorphs rather than alleles because they differ in sequence, size, and gene content (1). Characterization of homothallic and heterothallic strains of S. cerevisiae by Winge and Lindegren (2) led to the discovery of genetic loci controlling homothallism and ultimately to the cassette model of mating-type switching proposed by Herskowitz and colleagues (3). The cassette model, which has been thoroughly verified by experimentation (4, 5), states that a haploid cell can switch genotype at the MAT locus (from MATa to MAT␣ or vice versa) by a gene-conversion process that involves replacing the genetic information at MAT with information copied from one of two silent cassette loci, HML␣ or HMRa. The gene conversion is initiated at a double-stranded DNA break made by the Ho endonuclease, encoded by the HO gene on chromosome IV, which cuts at a site that marks the boundary between the Y sequences unique to the MATa or MAT␣ idiomorphs and the shared Z sequence flanking them. HO is expressed only in cells that have budded once, which means that only mother cells switch mating type and neighboring cells in a colony can mate (4). Hence, most natural isolates of S. cerevisiae are diploid and phenotypic...

“…1B). The maturase is a member of a large family of DNA endonucleases identifiable, in part, by conserved LAGLIDADG motifs (20). The presence of the bI3 maturase ORF within the intron probably represents an early colonization event by a LAGLI-DADG DNA endonuclease precursor (21)(22)(23)(24).…”

mentioning

confidence: 99%

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

Bassi

Oliveira

White

et al. 2002

Detectable splicing by the Saccharomyces cerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclearencoded protein, Mrs1. Both proteins bind independently to the bI3 RNA. The bI3 maturase binds as a monomer, whereas Mrs1 is a dimer in solution that assembles as two dimers, cooperatively, on the RNA. The active six-subunit complex has a molecular mass of 420 kDa, splices with a k cat of 0.3 min ؊1 , and binds the guanosine nucleophile with an affinity comparable to other group I introns. The functional bI3 maturase domain is translated from within the RNA that encodes the intron, has evolved a high-affinity RNAbinding activity, and is a member of the LAGLIDADG family of DNA endonucleases, but appears to have lost DNA cleavage activity. Mrs1 is a divergent member of the RNase H fold superfamily of dimeric DNA junction-resolving enzymes that also appears to have lost its nuclease activity and now functions as a tetramer in RNA binding. Thus, the bI3 ribonucleoprotein is the product of a process in which a once-catalytically active RNA now obligatorily requires two facilitating protein cofactors, both of which are compromised in their original functions. M any small ribozymes, both those that occur naturally (1) and those that are products of in vitro selection experiments (2), display an impressive array of catalytic activities. Although these simple structures are capable of performing catalysis, most cellular ribozymes have undergone a process of elaboration in which potentially simple RNA active sites are bolstered by additional RNA structural domains (3, 4) or protein cofactors (5-9).One example of this process is the group I introns. The group I intron RNA core consists of two extended and roughly coaxially stacked helices that form a catalytic cleft, which binds, in turn, the 5Ј and 3Ј splice site helices and the guanosine cofactor (guanosine 5Ј-monophosphate, pG) (4, 5, 10). Although the catalytic core is relatively compact and can independently form a functional active site (11), most group I introns have acquired additional peripheral structures that function to stabilize the RNA core (4, 12, 13). Moreover, whereas a large number of group I introns have been identified by sequence and structural analysis (4, 14), both anecdotal and experimental evidence suggests that many have lost their self-splicing activity (15). It appears that group I intron RNAs commonly recruit protein cofactors and now function as obligatory ribonucleoproteins (8, 16).The Saccharomyces cerevisiae mitochondrial cytochrome b bI3 group I intron represents an especially intriguing case of elaboration by accretion of peripheral RNA domains and protein cofactors. First, the bI3 RNA potentially spans two group I intron subgroups, IA2 and IB4, characterized by RNA sequence insertions between helices P7 and P3 and adjacent to P9 and by an extension of the P5 helix, respectively (4) (Fig. 1A, boxed structures). The bI3 intron also contains two tertiary inter...