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PubMed2003

Database for mobile group II introns.

Group II introns are self-splicing RNAs and retroelements found in bacteria and lower eukaryotic organelles. During the past several years, they have been uncovered in surprising numbers in bacteria due to the genome sequencing projects; however, most of the newly sequenced introns are not correctly identified. We have initiated an ongoing web site database for mobile group II introns in order to provide correct information on the introns, particularly in bacteria. Information in the web site includes: (1) introductory information on group II introns; (2) detailed information on subfamilies of intron RNA structures and intron-encoded proteins; (3) a listing of identified introns with correct boundaries, RNA secondary structures and other detailed information; and (4) phylogenetic and evolutionary information. The comparative data should facilitate study of the function, spread and evolution of group II introns. The database can be accessed at http://www.fp.ucalgary.ca/group2introns/.

Databases, Nucleic Acid↗

PubMed1995

Structure and activities of group II introns.

Group II introns are found in eubacteria and eubacterial-derived, organellar genomes. They have ribozymic activities, by which they direct and catalyze the splicing of the exons flanking them. This chapter reviews the secondary structure and known tertiary interactions of the ribozymic component of group II introns in relation to the problems of specifying splice sites and building a catalytic core. We pay special attention to the relationship between the transesterification and hydrolytic modes of initiating splicing and the stereospecificities of these reactions. A number of group II introns encode proteins of the reverse transcriptase family; the activity of these proteins enables the host introns to change genomic locations by mechanisms that are only beginning to be deciphered. Finally, we briefly discuss multipartite and post-transcriptionally edited group II introns, together with the intron microcosm of Euglena gracilis chloroplasts and the possible relationships between group II and spliceosome-catalyzed splicing processes.

Animals↗

PubMed2002

Identification and characterization of bacterial class E group II introns.

Group II introns are catalytic RNAs and mobile genetic elements. Phylogenetic characterization of group II intron-encoded reverse transcriptases (RTs) established seven classes: the mitochondrial class, chloroplast-like classes 1 and 2, and bacterial classes A, B, C, and D. In this study, we identified and characterized a new bacterial class of group II introns, bacterial class E, on the basis of phylogenetic analysis of the intron-encoded protein (IEP) RT and determination of a consensus intron RNA structure.

Bacteria↗

PubMed2000

A tertiary interaction that links active-site domains to the 5' splice site of a group II intron.

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.

Base Pairing↗

PubMed2000

Tri-partite assay for studying exon ligation by the ai5gamma group II intron.

Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).

Base Sequence↗

PubMed2001

The ins and outs of group II introns.

Group II introns have attracted considerable attention as ribozymes, mobile genetic elements and possible progenitors of nuclear spliceosomal introns. Major advances in understanding their catalytic structure and dispersal strategies have recently come from several model mitochondrial and bacterial self-splicing introns. In Nature, this family of introns shows wide variation in both features and behaviour, and this review includes a focus on the diversity of evolutionary pathways taken.

Animals↗

PubMed1993

Group I and group II introns.

Group I and group II introns are two types of RNA enzymes, ribozymes, that catalyze their own splicing by different mechanisms. In this review, we summarize current information about the structures of group I and group II introns, their RNA-catalyzed reactions, the facilitation of RNA-catalyzed splicing by protein factors, and the ability of the introns to function as mobile elements. The RNA-based enzymatic reactions and intron mobility provide a framework for considering the role of primordial catalytic RNAs in evolution and the origin of introns in higher organisms.

Amino Acid Sequence↗

PubMed1993

Interaction of intronic boundaries is required for the second splicing step efficiency of a group II intron.

Group II and nuclear pre-mRNAs introns share a common splicing pathway involving a lariat intermediate, as well as some primary sequence similarities at the splice junctions. In this work, we analyze the role of the conserved nucleotides at the first and penultimate positions (G1 and A886) of a group II self-splicing intron. We show that the G1 nucleotide is essential for the efficiency of both the first and the second splicing steps, while substitutions at the penultimate nucleotide affect mostly the efficiency of the second step. A reciprocal suppression of the second splicing step defect can be observed in some double mutants. This result is best explained by a non-Watson-Crick interaction between the first and the penultimate nucleotides of the intron, which occurs after lariat formation. The finding that an interaction between intron boundaries is required for the second splicing step in both group II and nuclear pre-mRNA introns strengthens the idea that both systems employ similar mechanisms, albeit with differences in the details of the nucleotide interactions.

Base Sequence↗

PubMed2004

Mobile group II introns.

Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements. They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage. Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns. We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity.

Bacteria↗

PubMed2001

Phylogenetic relationships among group II intron ORFs.

Group II introns are widely believed to have been ancestors of spliceosomal introns, yet little is known about their own evolutionary history. In order to address the evolution of mobile group II introns, we have compiled 71 open reading frames (ORFs) related to group II intron reverse transcriptases and subjected their derived amino acid sequences to phylogenetic analysis. The phylogenetic tree was rooted with reverse transcriptases (RTs) of non-long terminal repeat retroelements, and the inferred phylogeny reveals two major clusters which we term the mitochondrial and chloroplast-like lineages. Bacterial ORFs are mainly positioned at the bases of the two lineages but with weak bootstrap support. The data give an overview of an apparently high degree of horizontal transfer of group II intron ORFs, mostly among related organisms but also between organelles and bacteria. The Zn domain (nuclease) and YADD motif (RT active site) were lost multiple times during evolution. Differences in domain structures suggest that the oldest ORFs were concise, while the ORF in the mitochondrial lineage subsequently expanded in three locations. The data are consistent with a bacterial origin for mobile group II introns.

Amino Acid Sequence↗

PubMed2002

Productive folding to the native state by a group II intron ribozyme.

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.

Base Pairing↗

PubMed2001

Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis.

Group II introns require numerous divalent metal ions for folding and catalysis. However, because little information about individual metal ions exists, elucidating their ligands, functional roles and relationships to each other remains challenging. Here we provide evidence that an essential motif at the catalytic center of the group II intron, the AGC triad within domain 5 (D5), provides a ligand for a crucial metal ion. Sulfur substitution of the pro-Sp oxygen of the adenosine strongly disrupts D5 binding to a substrate consisting of an exon and domains 1-3 of the intron (exD123). Cd2+ rescues this effect by enabling the sulfur-modified D5 to bind to exD123 with wild type affinity and catalyze 5'-splice site cleavage. This switch in metal specificity implies that a metal ion interacts with D5 to mediate packing interactions with D123. This new D5 metal ion rescues the disruption of D5 binding and catalysis with a thermodynamic signature different from that of the metal ion that stabilizes the leaving group during the first step of splicing, suggesting the existence of two distinct metal ions.

Base Sequence↗

PubMed1999

A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA.

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.

Bacterial Proteins↗

PubMed2001

Visualizing the solvent-inaccessible core of a group II intron ribozyme.

Group II introns are well recognized for their remarkable catalytic capabilities, but little is known about their three-dimensional structures. In order to obtain a global view of an active enzyme, hydroxyl radical cleavage was used to define the solvent accessibility along the backbone of a ribozyme derived from group II intron ai5gamma. These studies show that a highly homogeneous ribozyme population folds into a catalytically compact structure with an extensively internalized catalytic core. In parallel, a model of the intron core was built based on known tertiary contacts. Although constructed independently of the footprinting data, the model implicates the same elements for involvement in the catalytic core of the intron.

Base Sequence↗

PubMed1992

Constraints on the evolution of plastid introns: the group II intron in the gene encoding tRNA-Val(UAC).

The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.

Base Sequence↗

PubMed1991

Structural requirements for selection of 5'- and 3' splice sites of group II introns.

The group II intron bl1 in the gene for apocytochrome b in yeast mitochondrial DNA (COB) is self-splicing in vitro. It could recently be shown that self-splicing of this intron is fully reversible in vitro. In addition, intron integration is not restricted to parental exons, since the intron can also integrate into a foreign RNA. The position of insertion seems to be immediately 3' to a cryptic intron binding site 1 (IBS1). We confirmed and extended these results by sequencing 26 individual RNAs with transposed introns after reverse transcription and PCR amplification. Results show that intron integration into authentic exons is generally correct, but that integration into a foreign RNA is often inaccurate, i.e. insertion is one nt downstream or upstream of the 3' end of IBS1. This leads to the generation of 5' splice junctions of the new intron-harbouring 'preRNAs' with addition (or deletion) of a single A residue at the 3' end of IBS1. To investigate which structures help to define the position of 5'- and 3' cleavage, preRNAs of i) these clones with aberrant 5' splice junctions and ii) preRNAs with artificial hairpins between domains 5 and 6 of the intron were spliced under different reaction conditions. Results obtained let us conclude that i) branchpoint dependent 5' cleavage is directed by the 5' terminal G residue of the intron and, ii) the first nucleotide(s) of the 3' exon play an important role in defining the 3' splice site.

Apoproteins↗

PubMed2003

The pathway for DNA recognition and RNA integration by a group II intron retrotransposon.

Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription.

Bacterial Proteins↗

PubMed2000

Metal ion binding sites in a group II intron core.

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.

Base Sequence↗