Organization of a ribosomal RNA gene cluster from the archaebacterium Methanothrix soehngenii.
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Archaea↗
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Results for “RNA, Transfer, Ala”
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Transcription of a silkworm tRNA(cAla) gene is directed by two AT-rich upstream sequence elements.
A region within 35 nucleotides upstream of the transcription initiation site of a variety of silkworm Class III templates is absolutely required for transcription in vitro. To determine whether the activity of this region can be attributed to a particular sequence element, we systematically replaced 4-5 bp segments of the region upstream of a silkworm tRNA(cAla) gene. We show that replacement of either of two AT-rich blocks markedly impairs promoter function, whereas replacement of other sequences has little or no effect. Additional mutants were constructed to test whether base composition or sequence is important for function of the AT blocks. We find that some sequences are more effective than others, but that various AT-rich sequences can direct transcription at a high level. Possible mechanisms by which such elements could act are discussed.
Animals↗
In vitro suppression as a tool for the investigation of translation initiation.
An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase. Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.
Amino Acid Sequence↗
Thermodynamics of RNA hairpins containing single internal mismatches.
Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions. Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities. The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site. UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins. Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability. In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities. The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed.
Circular Dichroism↗
Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond.
Single-stranded regions in RNA secondary structure are important for RNA-RNA and RNA-protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals 'well-determined' single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit beta-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA-RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.
Algorithms↗
Importance of the reverse Hoogsteen base pair 54-58 for tRNA function.
To elucidate the general constraints imposed on the structure of the D- and T-loops in functional tRNAs, active suppressor tRNAs were selected in vivo from a combinatorial tRNA gene library in which several nucleotide positions of these loops were randomized. Analysis of the nucleotide sequences of the selected clones demonstrates that among the randomized nucleotides, the most conservative are nucleotides 54 and 58 in the T-loop. In most cases, they make the combination U54-A58, which allows the formation of the normal reverse Hoogsteen base pair. Surprisingly, other clones have either the combination G54-A58 or G54-G58. However, molecular modeling shows that these purine-purine base pairs can very closely mimic the reverse Hoogsteen base pair U-A and thus can replace it in the T-loop of a functional tRNA. This places the reverse Hoogsteen base pair 54-58 as one of the most important structural aspects of tRNA functionality. We suggest that the major role of this base pair is to preserve the conformation of dinucleotide 59-60 and, through this, to maintain the general architecture of the tRNA L-form.
Anticodon↗
A class III transcription factor composed of RNA.
It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease.
Animals↗
Specificity for aminoacylation of an RNA helix: an unpaired, exocyclic amino group in the minor groove.
An acceptor stem G3.U70 base pair is a major determinant of the identity of an alanine transfer RNA. Hairpin helices and RNA duplexes consisting of complementary single strands are aminoacylated with alanine if they contain G3.U70. Chemical synthesis of RNA duplexes enabled the introduction of base analogs that tested the role of specific functional groups in the major and minor grooves of the RNA helix. The results of these experiments indicate that an unpaired guanine 2-amino group at a specific position in the minor groove of an RNA helix marks a molecule for aminoacylation with alanine.
Acylation↗
Identification of the sex chromosomes of the medaka, Oryzias latipes, by fluorescence in situ hybridization.
In the medaka, Oryzias latipes, which does not have cytologically recognizable sex chromosomes, the sex is genetically determined and the mechanism of sex determination (XX/XY) can be revealed by genetic crosses using a particular pigment gene. In a previous study, we isolated a sex-linked DNA marker (SL1) using the genomic differences between inbred strains of medaka. In the present paper, we further isolated another sex-linked clone (pHO5.110). The pHO5. 110-related sequences were tightly linked to sex in O. latipes. We designated the locus of the pHO5.110-related sequence on sex chromosomes of medaka as Sex-Linked 2 (SL2). Southern blot analyses suggested that the pHO5.110-related sequence was tandemly repetitive in the medaka genome. Using the clone as a probe for FISH analysis, strong hybridization signals were obtained in a couple of chromosomes that formed one of two large submetacentric chromosome pairs. The pHO5.110-related sequences were repetitive in the genomes of other species of Oryzias (O. curvinotus, O. luzonensis and O. mekongensis) that are karyologically related to O. latipes (all are members of the so-called biarmed group). By contrast, the sequences were not detected as repetitive in other Oryzias species. Hence, it is thought that pHO5.110-related sequences were amplified in the genome of a common ancestor of the biarmed group.
Animals↗
Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria.
BACKGROUND: Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNAala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNAala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNAala genes. RESULTS: The three tRNAala genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNAalaU and tRNAalaV containing the attB site, a single base difference was observed between the two species. We observed that the tRNAalaU gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNAalaV gene was used as the target. No integration occurred in the BCG tRNAalaU T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNAala T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNAalaU and tRNAalaV T-loops of the same BCG chromosome. CONCLUSION: Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNAala genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies.
Attachment Sites, Microbiological↗
Selective disruption of neuregulin-1 function in vertebrate embryos using ribozyme-tRNA transgenes.
The products of the neuregulin-1 gene constitute a set of polypeptide growth factors whose signalling through the ErbB receptors is essential to the growth and differentiation of many cell types in culture. Although studies with neuregulin-1 mutant mice have demonstrated that these growth factors are also essential regulators of cellular differentiation in vivo, the mid-embryonic death of these mutants precludes an analysis of hypothesized neuregulin-1 roles in later aspects of development. To circumvent this early lethality, we have pursued a ribozyme-based strategy for the perturbation of neuregulin-1 function in developing chick embryos. Early administration of a retrovirus carrying neuregulin-1 hammerhead-type ribozymes to blastoderm-stage embryos leads to an embryonic lethal phenotype that results from the failure of ventricular trabeculation in the developing heart, a faithful phenocopy of the mouse neuregulin-1 mutations. Later, more localized delivery of the ribozyme to the developing retina inhibits both the differentiation of retinal ganglion cell neurons and the proliferation of the neuroepithelial cells from which they derive. These results suggest that neuregulin-1 promotes both muscle cell differentiation in the heart and neuronal differentiation in the central nervous system. In addition, they demonstrate the utility of hammerhead ribozymes as a simple, effective and easily adaptable method of conditional gene inactivation in vertebrates.
Animals↗
Aminoacylation of alanine minihelices. "Discriminator" base modulates transition state of single turnover reaction.
RNA hairpin minihelices that recreate the acceptor-T psi C stem of Escherichia coli alanine tRNA are charged specifically with alanine, provided that they encode the critical G3:U70 base pair that is the major determinant for the identity of an alanine tRNA. These model substrates were used to investigate the role in charging of N73, the unpaired nucleotide that is just three positions removed from the amino acid attachment site and which is sometimes referred to as the "discriminator" base. Previous work showed that, while substrates which encode G3:U70 are all charged by alanine tRNA synthetase regardless of the base at position 73, catalytic efficiency is substantially higher with substrates that have the wild-type A73. To identify a specific step in aminoacylation that is affected by substitutions of A73, we studied the single turnover charging of A73, U73, C73, and G73 minihelices, using preformed, enzyme-bound alanyl adenylate and saturating concentrations of the respective minihelices. Compared to the A73 substrate, the transfer of activated amino acid to bound RNA is sharply reduced for the substituted N73 minihelices. The low efficiency of transfer is not due to an abortive reaction in which the adenylate or a transiently charged RNA is hydrolyzed. Instead, under the conditions used, the active adenylate remains on the enzyme for extended periods and simply reacts slowly with bound N73 RNA. The results suggest that the nature of the discriminator base is a critical determinant of the transition state for the reaction of bound alanyl adenylate with RNA on the surface of the enzyme.
Acylation↗
Cloning of DNA fragments containing alanine tRNA sequences from a spider genome.
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Animals↗
Rationale for engineering an enzyme by introducing a mutation that compensates for a deletion.
Procedures are needed for the manipulation of enzymes to introduce improved catalytic efficiencies and substrate affinities, even in the absence of information on three-dimensional structure. We previously described a selection for amino acid replacements that compensate for a large polypeptide deletion in an enzyme. The rationale is that, because of the missing polypeptide sequence, compensatory replacements must create new enzyme-substrate contacts that are not present in the native protein. Here we introduce into an undeleted enzyme a mutation that compensates for a large deletion in the same enzyme. Binding and kinetic measurements show that this mutation has the same effect on the undeleted as on the deleted protein. The results suggest that a new enzyme-substrate interaction has been created by the mutation and that its contribution to the interaction energy is similar in both the deleted and undeleted molecules. Introduction of this class of mutations into enzymes may in general be useful for engineering them to have improved interactions with ligands and substrates.
Alanine-tRNA Ligase↗
Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.
The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.
Bacterial Proteins↗
[Analysis of autoantigens and clinical significance of antinuclear antibodies].
A highly sensitive RNA-immunoprecipitation assay (Lerner-Steitz assay) is a unique and useful method of identifying autoantibodies to RNA-associated antigens. In this study, we identified novel autoantibodies to tRNAs using RNA-immunoprecipitation assay. In screening of 1472 sera by RNA-immunoprecipitation using HeLa cell extracts as an antigen source, 41 sera were found to immunoprecipitate tRNAs. Fifteen of these 41 sera also immunoprecipitated deproteinized tRNAs, indicating that these 15 sera contained anti-tRNA antibodies. Three sera immunoprecipitated naked tRNA from E. coli. When in vitro transcripts from cDNAs encoding E. coli tRNAs and their synthesized mutants were used as antigens, it was demonstrated that the representative serum recognized the conformational epitope of the "L"-shape structure which was conserved in all tRNAs of all species. This finding suggests the role of bacterial infection in the development of autoantibodies and autoimmune diseases. Two of 15 sera containing anti-tRNA antibodies were identified as anti-PL-12 (alanyl-tRNA synthetase) antibodies. Eleven of the remaining 13 patients were diagnosed as either SLE, Sjögren's syndrome or their overlap. In addition, fever, Raynaud's phenomenon, polyarthritis, leukocytopenia and characteristic annular erythema were frequently found in these patients. Novel autoantibodies to tRNAs appeared to be associated with common clinical features but were distinct from previously described anti-aminoacyl-tRNA synthetases.
Alanine-tRNA Ligase↗
Inter- and intraspecies variations of the 16S-23S rDNA intergenic spacer region of various streptococcal species.
The 16S-23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. uberis, S. parauberis, S. pyogenes and S. equi subsp. equi, respectively. The amplicons of S. equi subsp. zooepidemicus, S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. pyogenes and S. suis, 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi, S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA(Ala)), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus, S. porcinus and S. suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.
DNA, Bacterial↗
Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis.
Euglena gracilis cells synthesize the key tetrapyrrole precursor, delta-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain alpha-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, alpha-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and alpha-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP(+), and was inhibited by NAD(+). Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of alpha-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.
Journal Article↗