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PubMed1992

Intervening sequences in an Archaea DNA polymerase gene.

The DNA polymerase gene from the Archaea Thermococcus litoralis has been cloned and expressed in Escherichia coli. It is split by two intervening sequences (IVSs) that form one continuous open reading frame with the three polymerase exons. To our knowledge, neither IVS is similar to previously described introns. However, the deduced amino acid sequences of both IVSs are similar to open reading frames present in mobile group I introns. The second IVS (IVS2) encodes an endonuclease, I-Tli I, that cleaves at the exon 2-exon 3 junction after IVS2 has been deleted. IVS2 self-splices in E. coli to yield active polymerase, but processing is abolished if the IVS2 reading frame is disrupted. Silent changes in the DNA sequence at the exon 2-IVS2 junction that maintain the original protein sequence do not inhibit splicing. These data suggest that protein rather than mRNA splicing may be responsible for production of the mature polymerase.

Amino Acid Sequence↗
PubMed1990

Group II intron RNA-catalyzed recombination of RNA in vitro.

We report the first evidence for a novel reaction mediated by the self-splicing yeast mitochondrial group II intron bl1; the site-specific recombination of RNA molecules in vitro. Upon incubation of the intron lariat with two different RNAs, each harbouring a short sequence complementary to exon binding site 1 (EBS1) of the intron, novel recombined RNAs are formed. As a result of this intron-mediated shuffling of gene segments, the 5' part of RNA1 is ligated to the 3' part of RNA2 and, reciprocally, the 5' part of RNA2 to the 3' part of RNA1. Sequence analysis of the recombinant junction shows that the site of recombination is precisely located 3' to intron binding site 1 (IBS1). The hypothesized mechanism of recombination involves exchange of RNA 5' parts after the first step of a reverse splicing reaction. The possible role of this mechanism in vivo and during prebiotic evolution is discussed.

Base Sequence↗
PubMed1991

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron.

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.

Animals↗
PubMed1992

Sequence and variability of the 5.8S and 26S rRNA genes of Pneumocystis carinii.

The sequence of the coding region of the rRNA operon of rat-derived Pneumocystis carinii has been completed, including the genes for 5.8S and 26S rRNA. These genes show homology to the rRNA genes of yeast, and an apparent group I self-splicing intron is present in the 26S rRNA gene. Like a similar intron in the 16S rRNA gene, this intron is in a phylogenetically conserved region. Variation in the 26S rRNA sequence was noted between P. carinii organisms isolated from two different sources.

Base Sequence↗
PubMed1992

Protein splicing removes intervening sequences in an archaea DNA polymerase.

The Vent DNA polymerase gene from Thermococcus litoralis contains two in-frame insertions that must be spliced out to form the mature polymerase. Primer extension and cDNA PCR revealed no evidence of spliced RNA to account for this editing. In contrast, pulse-chase analysis indicated that expression constructs lacking the first insertion produced a protein precursor in Escherichia coli that was processed post-translationally to form polymerase and I-TliI, the endonuclease protein that is the product of the second insertion. At least one intermediate, which migrated more slowly than the precursor and may be branched, was also detected. Amino acid substitutions at the splice junction slowed or blocked the protein splicing reaction. Processing occurs in several heterologous systems, indicating either self-splicing or ubiquitous splicing factors. Processing occurs in a mutant lacking I-TliI endonuclease activity, establishing the independence of splicing and endonuclease activities.

Amino Acid Sequence↗
PubMed1992

Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis.

Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391-395). We studied the effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 x 10(3) when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 x 10(5). The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. In addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.

Animals↗
PubMed1990

Mechanism of 3' splice site selection by the catalytic core of the sunY intron of bacteriophage T4: the role of a novel base-pairing interaction in group I introns.

The catalytic core of the sunY intron of bacteriophage T4 is separated from its 3' exon by 837 nucleotides, most of which are part of an open reading frame (ORF). Here, we report that transcripts truncated within the sunY ORF self-splice in vitro to a variety of sites in the segment immediately 3' of the core. Recognition of these proximal splice sites is shown to depend on (1) the presence on the intron side of a terminal G, which must not be part of a secondary structure; and (2) the ability of the penultimate intron nucleotide to base-pair with a 3' splice site-binding sequence (3'SSBS) located within the core. The counterpart of the 3'SSBS can be identified in most group I introns. The possible significance of such alternative splicing events for in vivo expression of intron-encoded proteins is discussed.

Base Composition↗
PubMed1991

The phage T4 nrdB intron: a deletion mutant of a version found in the wild.

Bacteriophage T4 possesses three self-splicing group I introns. Two of the three introns are mobile elements; the third, in the gene encoding a subunit of the phage nucleotide reductase (nrdB), is not mobile. Because intron mobility offers a reasonable explanation for the paradoxical occurrence of large intervening sequences in a space-efficient eubacterial phage, it is puzzling that the nrdB intron is not mobile like its compatriots. We have discovered a larger nrdB intron in a closely related phage, and we infer from comparative sequence data that the T4 intron is a deletion mutant derived from this larger intron. This larger nrdB intron encodes an open reading frame of 269 codons, which we have cloned and overexpressed. The overexpressed protein shows a dsDNA endonuclease activity specific for the intronless nrdB gene, typical of mobile introns. Thus, we believe that all three introns of T4 are or were mobile "infectious introns" and that they have entered into and been maintained in the phage population by virtue of this efficient mobility.

Amino Acid Sequence↗
PubMed1992

Activation of the catalytic core of a group I intron by a remote 3' splice junction.

Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions. Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo. Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction. Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation. A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing. A role for these interactions in mediating putative alternative splicing events is discussed.

Base Sequence↗
PubMed1992

Selection of a ribozyme that functions as a superior template in a self-copying reaction.

The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.

Bacteriophage T4↗
PubMed1992

Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites.

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.

Animals↗
PubMed1991

A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis.

Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

Animals↗
PubMed1991

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein.

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.

Adenosine Triphosphate↗
PubMed1991

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Animals↗
PubMed1991

The product of the split sunY gene of bacteriophage T4 is a processed protein.

The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 amino acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.

Genes, Viral↗
PubMed1990

Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.

Amino Acid Sequence↗
PubMed1992

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.

Amino Acid Sequence↗
PubMedDate not supplied

[Design of ribozymes].

A review of the data on the synthesis of RNA-fragments with endonuclease activity. The Haseloff and Gerlach model for design of ribozymes and the results of the synthesis of the three new ribozymes targeted against the chloramphenicol acetyl transferase gene transcript are considered. The possibility of the use of a shortened form of Tetrahymena self-splicing ribosomal RNA intervening sequence for the ribozyme design also are discussed.

Base Sequence↗