Base Pairs In Stem Research Articles

A novel mutation in the mitochondrial tRNA Phe gene associated with mitochondrial myopathy

We report a novel heteroplasmic T→C mutation at nt position 582 within the mitochondrial tRNA Phe gene of a 70-year-old woman with mitochondrial myopathy. No other family members were affected, suggesting that our patient was a sporadic case. The muscle showed frequent ragged red fibers and 43% cytochrome c oxidase deficient fibers. The mutation alters a conserved base pairing in the aminoacyl acceptor stem. The mutation load was 70% in muscle homogenate and varied from 0 to 95% in individual muscle fiber segments. Cytochrome c oxidase-negative fibers showed significantly higher levels of mutated mtDNA (>75%) than Cytochrome c oxidase-positive fibers (<55%). This mutation adds to the previously described four pathogenic mutations in the tRNA Phe gene.

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone.

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.

Early onset of rejection in concordant hamster xeno hearts display signs of necrosis, but not apoptosis, correlating to the phosphocreatine concentration

Background: The importance of apoptosis contra necrosis for ischemia/reperfusion (RP) and acute rejection in concordant rodent xenotransplantation is largely unknown. We explored this question by comparing rodent allo and concordant xenotransplants with different morphological methods to detect apoptosis and biochemical data on the levels of high-energy phosphates obtained with in vitro 31Phosphorous Magnetic Resonance Spectroscopy ( 31P MRS). More specifically, we applied a hitherto unused method in transplantation research, apoptosis specific biotin labeled oligonucleotides designed with a 10 base pair stem region and a 20 nucleotides large loop that form a hairpin like shape. The results obtained with this method were compared to results obtained with the more widely used in situ 3′-end labeling of DNA (TUNEL) assay and extraction and gel electrophoresis of labeled DNA (DNA laddering). Methods: Cervical heart transplantations were performed between inbred Lewis (L) (RT1 1) to L, L to DA (RT1 a) rats, hamster (H) to H and H to L (X) ( n=5 for all groups except for X, n=9). All hearts were subjected to 30 min of cold ischemia (+4 °C) and 6 h of RP before explantation. In vitro 31P MRS was used to determine the phosphocreatine (PCr), beta-adenosine triphosphate (β-ATP) concentrations and the PCr/β-ATP ratio of the transplants. We correlated the biochemical data to haematoxylin and eosin (H & E) stained tissue slides scored for rejection, infiltration of antibodies and complement depositions, DNA extraction and gel electrophoresis of labeled DNA (DNA laddering), in situ 3′-end labeling of DNA (TUNEL) and the apoptosis specific hairpin probe assays scoring. Results: The rejection score of the xeno grafts differed significantly compared to their syngeneic hamster to hamster controls (2.40±0.25 vs. 1.20±0.20; P=0.005) and they had a significantly higher TUNEL score, 228±15 vs. 2.44±0.32 ( P=0.009), that correlated to changes in PCr concentration ( P<0.001) and to the PCr/β-ATP ratio ( P=0.01). The uptake was mainly (90–95%) located to 1–2 μm large extra cellular ‘granule’. A picture resembling early necrosis was seen on the H & E stainings and reflected in the Billingham rejection score above. Conclusions: After 6 h of RP the onset of acute rejection in the concordant hamster xeno hearts displayed features of early, possibly mitochondrial, necrosis, but not apoptosis, which correlated to changes in the PCr concentration and the PCr/β-ATP ratio. The mechanism for the early rejection observed is unclear and might be caused by other factors in the sera apart from cellular components, antibodies and complement factors. Identification of the underlying mechanisms could enable us to design rational therapies that prevent activation of the recipient's innate immune response.

Identification of Cleavage Sites in the HIV-1 TAR RNA by 10-23 and 8-17 Catalytic Motif Containing DNA Enzymes

A quick identification of a cleavage site in the target RNA molecule to obtain sequence-specific cleavage by either catalytic RNA (ribozymes) or DNA (DNA enzymes) is very important for achieving gene-specific suppression. These molecules could also provide important information on the secondary and tertiary structure of the target RNA molecule. We have exploited the use of two kinds of DNA enzymes, namely, the 10-23 and 8-17 catalytic motif containing DNA enzymes, to achieve these objectives. We identified several DNA enzyme cleavage sites in the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) RNA-a structural feature present at the 5' end of all HIV-1 transcripts. Most of the DNA enzymes that cleaved the TAR RNA were targeted to the regions that were single-stranded in the predicted structure. Regions that were predicted to be base-paired (stem) failed to show any detectable cleavage. The DNA enzyme possessing the 8-17 catalytic motif was extremely efficient in cleaving full length, as well as short, HIV-1 specific transcripts. The efficiency of cleavage of the same target RNA by DNA enzymes that possessed the 10-23 catalytic motif was significantly less in comparison, and they failed to cleave the short transcripts. These molecules, in principle, have the potential to down regulate expression of all HIV-1 transcripts from a wide range of isolates because this region is functionally very well conserved.

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove.

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.

Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II.

Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg(2+) concentrations were in vitro selected from a library with 18 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had the same sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mM Mg(2+) concentration in the context of several substrates with differences in the lengths of stem I and III, including the well-characterised HH16 substrate and a derivative thereof with a GUA triplet at the cleavage site, as well as a substrate used previously in a related study. For the HH16 substrate, a change of base pair 10.2-11.2 to C-G in stem II further improved activity by about 2.5-fold to 0.8 min(-1) (at 1 mM Mg(2+) concentration, 25 degrees C, pH 7.5). Interestingly, this very active variant was not identified by the selection procedure. Changing loop II from UUUU to GCAA or extension of stem II to three or four base pairs reduced the cleavage rate by 2.0-2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so far at low Mg(2+) concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degrees C and pH 7.5 in monophasic reactions, with endpoints between 75 and 90 %. Such constructs promise to be advantageous for the inhibition of gene expression in vivo.

RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop

Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5′-GGGC[GCAA]GCCU-3′ were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type→S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2′-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.

Pseudouridylation of U 35 in the anticodon of Arabidopsis thaliana pre-tRNA Tyr depends on length rather than structure of an intron

In order to establish the structure and sequence requirements for pseudouridine (Ψ 35) biosynthesis in Arabidopsis thaliana tRNA Tyr five mutants of nuclear pre-tRNA Tyr have been prepared and analyzed: ΔI-tRNA Tyr transcript depleted of an intron, and 5UI, 7UI, 9UI and 12UI transcripts containing tracts of five, seven, nine and 12 U residues, respectively, instead of the wild type tRNA Tyr intron. The in vitro transcripts were incubated in a lupin seed extract containing Ψ 35 synthase activity, and those containing an artificial intron composed of 12 or nine U residues turned out to be good substrates for Ψ 35 synthase. The transcript with an intron composed of seven uridine residues was pseudouridylated up to 40%, whereas the remaining two were not pseudouridylated at all. The secondary structures of all transcripts were determined using enzymatic and chemical probes: S 1, V 1, T 1, A, P 1 and Pb 2+. All mutant pre-tRNAs show similar structural features: their anticodon arm contains a five base pair stem and a large loop which consists of five natural tRNA Tyr AC loop nucleotides to which five, seven, nine and 12 U residues are added. As the structure of the wild type pre-tRNA Tyr is different we propose that the role of its intron in the process of U 35 pseudouridylation is simply to expand the anticodon region to the required critical length.

Additional Hydrogen Bonds and Base-Pair Kinetics in the Symmetrical AMP-DNA Aptamer Complex

The solution structure of an adenosine monophosphate (AMP)-DNA aptamer complex has been determined previously [Lin, C. H., and Patel, D.J. (1997) Chem. Biol. 4:817–832]. On a symmetrical aptamer complex containing the same binding loop, but with better resolved spectra, we have identified two additional hydrogen bond-mediated associations in the binding loop. One of these involves a rapidly exchanging G imino proton. The phosphate group of the AMP ligand was identified as the acceptor by comparison with other aptamer complexes. Imino proton exchange measurements also yielded the dissociation constants of the stem and binding loop base pairs. This study shows that nuclear magnetic resonance-based imino proton exchange is a good probe for detection of weak hydrogen-bond associations.

Initiation of HIV-1 Reverse Transcription Is Regulated by a Primer Activation Signal

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome appears to be strictly regulated at the level of initiation. The primer binding site (PBS), at which the tRNA(3)(Lys) molecule anneals and reverse transcription is initiated, is present in a highly structured region of the untranslated leader RNA. Detailed mutational analysis of the U5 leader stem identified a sequence motif in the U5 region that is critical for activation of the PBS-bound tRNA(3)(Lys) primer. This U5 motif, termed the primer activation signal (PAS), may interact with the TPsiC arm of the tRNA(3)(Lys) primer, similar to the additional interaction proposed for the genome of Rous sarcoma virus and its tRNA(Trp) primer. This suggests that reverse transcription is regulated by a common mechanism in all retroviruses. In HIV-1, the PAS is masked through base pairing in the U5 leader stem. This provides a mechanism for positive and negative regulation of reverse transcription. Based on structure probing of the mutant and wild-type RNAs, an RNA secondary structure model is proposed that juxtaposes the critical PAS and PBS motifs.

Structure of an RNA hairpin from HRV-14.

The 5' noncoding region of the picornaviral genome begins with a cloverleaf which is required for viral replication, due at least in part to an interaction with the viral RNA polymerase as part of a fusion with the predominant viral protease. The necessary region of the cloverleaf has previously been narrowed to a highly conserved stem-loop. The solution structure of a 14-nucleotide RNA hairpin, which is part of the conserved stem-loop from human rhinovirus isotype 14, is presented here. The secondary structure of the hairpin is identical to predictions: a five base pair stem is bounded by a triloop with sequence UAU. However, the fold of the triloop is novel, with stacking of the second loop base onto the closing base pair of the stem, and deviations from A form geometry are introduced into the stem regions bordering the triloop, particularly on the 3' side. These deviations and the associated triloop structure could help to explain the distinct sequence conservation and mutational analysis data observed for the stem region of the hairpin, as compared to a second sequentially similar stem in the intact stem-loop.

Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs

Ro RNPs are small cytoplasmic RNA-protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264-273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5'/3' terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122-10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5'/3' double-stranded stem region that is conserved between different Y RNAs.

Structural and thermodynamic studies on mutant RNA motifs that impair the specificity between a viral replicase and its promoter

The 3′-end region of the genomic RNA of brome mosaic virus forms a tRNA-like structure that is critical for its replication. Previous studies have shown that in this region, a stem-loop structure, called SLC, is necessary and sufficient for the binding of the RNA replicase, and for RNA replication. Recently, we determined the high-resolution NMR structure of SLC, which demonstrated that a 5′-AUA-3′ triloop region is an important structural element for the enzymatic recognition. We proposed that the 5′-adenine of the triloop, which is rigidly fixed (“clamped”) to the stem, is a key recognition element for the replicase. To elucidate the role of this “clamped base motif” for the enzymatic recognition, we have now investigated the solution conformations of several stem-loop molecules with mutant triloops, 5′-UUA-3′, 5′-GUA-3′, 5′-CUA-3′ and 5′-UUU-3′, that destroy the enzymatic recognition. For the GUA and UUA mutants, we have obtained high-resolution solution structures using 2D NMR. All four mutants have very similar thermodynamic stabilities, and all have the same secondary structures, a triloop with a five base-paired stem helix. In addition, they have quite similar sugar puckering patterns in the triloop region. The NMR structures of the GUA and UUA show that the 5′ nucleotide of the triloop (G6 in GUA or U6 in UUA) lacks the strong interactions that hold its base in a fixed position. In particular, the U6 of UUA is found in two different conformations. Neither of these two mutants has the clamped base motif that was observed in the wild-type. While UUA also shows global change in the overall triloop conformation, GUA shows a very similar triloop conformation to the wild-type except for the lack of this motif. The absence of the clamped base motif is the only common structural difference between these two mutants and the wild-type. These results clearly indicate that the loss of function of the UUA and GUA mutants comes mainly from the destruction of a small key recognition motif rather than from global changes in their triloop conformations. Based on this study, we conclude that the key structural motif in the triloop recognized by the replicase is a solution-exposed, 5′-adenine base in the triloop that is clamped to the stem helix, which is called a clamped adenine motif.

Base-pair alterations in the epsilon-lower stem due to a novel double substitution in the precore gene of HBV-e negative variant were recovered by secondary mutations.

The HBe negative phenotype, a natural precore mutant (G1896A/G1897A) of HBV with aborted HBeAg expression is known to cause chronic hepatitis. The destabilized C : G base-pairing in the lower stem of epsilon-hairpin due to G1896A substitution is reportedly compensated by a second C1858T mutation and suggested to play an important role in enhanced selection of the HBe negative variant. We undertook to investigate presence of such compensatory mutations at other positions by analyzing epsilon-sequences (nts. 1847-1907) as well as to look for their effect(s), if any, on the consensus sequence of the overlapping core-initiator of HBe negative HBV variants in CLD patients. Three of the 5 HBe negative patients had classical G1896A mutation having a second compensatory mutation at nt. 1858. One patient showed an additional G1897A substitution, presenting as a novel precore stop codon mutation (UGG-->UAA), followed by a compensatory mutation at position 1857. In the third patient, a G1899A substitution was seen which compensated the impaired U at position 1855. Other substitution and deletion mutations were also observed in the remaining epsilon-hairpin, which however, did not produce any compensatory mutation. Further, all the precore variants showed a conserved G at position 1904, important for the optimal context of their core-initiator which however, remained impaired with A (nt. 1850). Our results suggest that the nts. 1851-1859 and nts. 1895-1904 in the lower stem, and restoration of authentic base-pairings therein, maintain the structural integrity and stability of the epsilon-hairpin. This may have a role in the enhanced selection of the HBe negative variants and persistence of HBV infection in chronic liver disease patients.

Stabilization of the U5-leader stem in the HIV-1 RNA genome affects initiation and elongation of reverse transcription.

Reverse transcription of the Human Immunodeficiency Virus type I (HIV-1) RNA genome is primed by a cellular tRNA-lys3 molecule that binds to the primer binding site (PBS). The PBS is predicted to be part of an extended RNA structure, consisting of a small U5-PBS hairpin and a large U5-leader stem. In this study we stabilized the U5-leader stem of HIV-1 to study its role in reverse transcription. We tested in vitro synthesized wild-type and mutant templates in primer annealing, initiation and elongation assays. Stabilization of the stem inhibits the initiation of reverse transcription, but not the annealing of the tRNA primer onto the PBS. These results suggest that stabilization of the stem results in occlusion of a sequence motif that is involved in an additional interaction with the tRNA-lys3 primer and that is needed to trigger the initiation of reverse transcription. The stable structure was also found to affect the elongation of reverse transcription, causing the RT enzyme to pause upon copying 7-8 bases into the extended base paired stem. The stabilizing mutations were also introduced into proviral constructs for replication studies, demonstrating that the mutant viruses have a reduced replication capacity. Analysis of a revertant virus demonstrated that opening of the stabilized U5-leader stem can restore both virus replication and reverse transcription.

TRNA leucine identity and recognition sets

Transfer RNAs (tRNAs) are grouped into two classes based on the structure of their variable loop. In Escherichia coli, tRNAs from three isoaccepting groups are classified as type II. Leucine tRNAs comprise one such group. We used both in vivo and in vitro approaches to determine the nucleotides that are required for tRNALeu function. In addition, to investigate the role of the tRNA fold, we compared the in vivo and in vitro characteristics of type I tRNALeu variants with their type II counterparts.A minimum of six conserved tRNALeu nucleotides were required to change the amino acid identity and recognition of a type II tRNASer amber suppressor from a serine to a leucine residue. Five of these nucleotides affect tRNA tertiary structure; the G15-C48 tertiary “Levitt base-pair” in tRNASer was changed to A15-U48; the number of nucleotides in the α and β regions of the D-loop was changed to achieve the positioning of G18 and G19 that is found in all tRNALeu; a base was inserted at position 47n between the base-paired extra stem and the T-stem; in addition the G73 “discriminator” base of tRNASer was changed to A73. This minimally altered tRNASer exclusively inserted leucine residues and was an excellent in vitro substrate for LeuRS. In a parallel experiment, nucleotide substitutions were made in a glutamine-inserting type I tRNA (RNASerΔ; an amber suppressor in which the tRNASer type II extra-stem-loop is replaced by a consensus type I loop). This “type I” swap experiment was successful both in vivo and in vitro but required more nucleotide substitutions than did the type II swap.The type I and II swaps revealed differences in the contributions of the tRNALeu acceptor stem base-pairs to tRNALeu function: in the type I, but not the type II fold, leucine specificity was contingent on the presence of the tRNALeu acceptor stem sequence. The type I and II tRNAs used in this study differed only in the sequence and structure of the variable loop. By altering this loop, and thereby possibly introducing subtle changes into the overall tRNA fold, it became possible to detect otherwise cryptic contributions of the acceptor stem sequence to recognition by LeuRS. Possible reasons for this effect are discussed.

DNA uptake signal sequences in naturally transformable bacteria

The naturally transformable bacterium Haemophilus influenzae Rd contains 1471 copies of the DNA uptake signal sequence (USS) 5'-AAGTGCGGT in its genome. Neisseria meningitidis contains 1891 copies of the USS sequence 5'-GCCGTCTGAA. The USSs are often found in the base paired stem of transcription terminators.

Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.

Selection of cleavage site by mammalian tRNA 3′ processing endoribonuclease

Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) removes 3′ trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3′ tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNA Arg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3′ trailer are sufficient for recognition by 3′ tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp ( N is less than 12) were cleaved 12— N and 13— N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3′ tRNase.

Designing a Novel Molecular Beacon for Surface-Immobilized DNA Hybridization Studies

Designing a Novel Molecular Beacon for Surface-Immobilized DNA Hybridization Studies