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PubMed2000

Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single-stranded RNAs.

A novel translation, trans-translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans-translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional.

Acylation↗
PubMed1992

Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during translation.

The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed at the alpha-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the epsilon-amino group of natural lysyl-tRNA, and each was used to nonenzymatically initiate peptide synthesis. The fluorescent alanyl-tRNAs containing an AAA anticodon were used to initiate polyserine (with a synthetic tRNA(Ser] or polyalanine synthesis from a poly(uridylic acid) template. The fluorescent lysyl-tRNA was used to initiate polylysine synthesis from poly(adenylic acid). Changes in the fluorescence of the amino-terminal coumarin were examined to characterize the environment of the probe as the nascent peptides were extended. Protection from proteolysis and the binding of anti-coumarin antibodies or Fab fragments suggest that the amino terminus of each polypeptide is protected from interaction with proteins (Mr greater than 28,000) until the peptides are extended to an average length of 40-50 residues; however, the fluorescence from the amino terminus of shorter nascent polyalanine and polyserine peptides was readily quenched by methyl viologen (Mr = 257), indicating ribosomes do not shield the nascent peptide from molecules of this size. The data appear to indicate that polyalanine, polyserine, and polylysine are extended from the peptidyl transferase into a protected region of the ribosome such as a groove or tunnel but that this region is readily accessible to small molecules.

Amino Acid Sequence↗
PubMed1992

Novel transfer RNAs that are active in Escherichia coli.

Many of the mammalian mitochondrial tRNAs contain significant nucleotide deletions in the dihydrouridine (D) stem or T psi C stem, so that they cannot fold into the canonical cloverleaf structure. This suggests that alternative forms and shapes are possible for a mitochondrial tRNA that functions in the specialized translational apparatus of the mammalian mitochondria. The question of whether significant structural alterations may be accommodated by a bacterial protein synthesis machinery, such as in Escherichia coli, is unanswered. In this work, all but ten positions in the gene for the 76-nucleotide coding sequence of an E. coli amber suppressor tRNA were permuted and screened for biological activity in vivo. Sequence analysis of a collection of biologically active variants established that many have unusual structures that include base-pair mismatches in helical stems, substitutions of normally conserved bases, and deletions. Independent mutations were obtained that weaken base pairs or tertiary interactions that normally stabilize the coaxial stacking of the D and anticodon stems, suggesting that the translational apparatus can accommodate considerable flexibility in this part of the molecule. The results demonstrate the capacity of the bacterial protein synthetic apparatus to accommodate altered tRNA structures that are not represented by any naturally occurring tRNAs.

Base Sequence↗
PubMed1992

Functional compensation of a recognition-defective transfer RNA by a distal base pair substitution.

A single G3:U70 base pair in the acceptor helix is the major determinant of alanine acceptance in alanine transfer RNAs. Transfer of this base pair into other transfer RNAs confers alanine acceptance. A G3:C70 substitution eliminates alanine acceptance in vivo and in vitro. In this work, a population of mutagenized G3:C70 alanine tRNA amber suppressors was subjected to a selection for mutations that compensate for the inactivating G3:C70 substitution. No compensatory mutations located in the acceptor helix were obtained. Instead, a U27:U43 substitution that replaced the wild-type C27:G43 in the anticodon stem created a U27:U43/G3:C70 mutant alanine tRNA that inserts alanine at amber codons in vivo. The U27:U43 substitution is at a location where previous footprinting work established an RNA-protein contact. Thus, this mutation may act by functionally coupling a distal part of the tRNA structure to the active site.

Amino Acid Sequence↗
PubMed2002

Poly(C) synthesis by class I and class II CCA-adding enzymes.

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.

Adenosine Triphosphate↗
PubMed2006

Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration.

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.

Acetylation↗
PubMed1991

Enzymatic aminoacylation of single-stranded RNA with an RNA cofactor.

A chemically synthesized single-stranded ribonucleotide tridecamer derived from the 3' end of Escherichia coli alanine tRNA can be charged with alanine in the presence of short complementary RNA oligonucleotides that form duplexes with the 3' fragment. Complementary 5' oligomers of 9, 8, 6, and 4 nucleotides all confer charging of the 3' fragment. Furthermore, in the presence of limiting 5' oligomer, greater than stoichiometric amounts of the single-stranded 3' acceptor fragment can be aminoacylated. This is due to a reiterative process of transient duplex formation followed by charging, dissociation of the 5' oligomer, and then rebinding to an uncharged single-stranded ribotridecamer so as to create another transient duplex substrate. Thus, a short RNA oligomer serves as a cofactor for a charging enzyme, and it thereby makes possible the aminoacylation of single-stranded RNA. These results expand possibilities for flexible routes to the development of early charging and coding systems.

Alanine-tRNA Ligase↗
PubMed1993

Neural BC1 RNA as an evolutionary marker: guinea pig remains a rodent.

The traditional morphologically grounded placement of South American guinea pig-like rodents (Caviomorpha) within one of the two rodent suborders, Hystricognathi, has been disputed by recent analysis of protein and nucleic acid sequence data. The Caviomorpha and possibly all Hystricognathi would be considered a separate order, distinct from the other rodent suborder, Sciurognathi, and thus of the order Rodentia, and would be placed closer phylogenetically to other mammals [Graur, D., Hide, W. A. & Li, W.-H. (1991) Nature (London) 351, 649-652]. To address the discrepancy between morphological comparisons and sequence analyses, we have applied an alternative form of molecular analysis. We demonstrate that BC1 RNA, a neural-specific small cytoplasmic RNA that is the product of a retropositionally generated gene (a gene derived by reverse transcription of RNA followed by insertion of the DNA copy into the genome), is present in Sciurognathi and guinea pig but not in other mammalian orders including Lagomorpha, Artiodactyla, and Primates. The species-confined, tissue-specific expression of a retroposed sequence therefore supports the morphological evidence for monophyly of Rodentia inclusive of guinea pig and demonstrates the usefulness of such molecular genetic markers. Furthermore, the conservation and tissue-specific expression of the BC1 RNA gene in the two divergent rodent suborders suggests that this macromolecule has been exapted into a functional role (i.e., coopted into a variant or novel function) in the rodent nervous system.

Animals↗
PubMed1997

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase.

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G.U base pair at position 3.70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I.U, G.C, and A.U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G.U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G. U with mismatches such as G.A and C.A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G.U pair. Although most of these substrates have undetectable activity, G.A and C.A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3.70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.

Alanine-tRNA Ligase↗
PubMed1999

Correlation of deformability at a tRNA recognition site and aminoacylation specificity.

The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G.U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G.U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G.U RNA, whereas inactive Watson-Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G.U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.

Acylation↗
PubMed1999

A TATA element is required for tRNA promoter activity and confers TATA-binding protein responsiveness in Drosophila Schneider-2 cells.

In contrast to yeast and mammalian systems, which depend principally on internal promoter elements for tRNA gene transcription, insect systems require additional upstream sequences. To understand the function of the upstream sequences, we have asked whether the Bombyx mori tRNACAla and tRNASGAla genes, which are absolutely dependent on these sequences in vitro, also require them for transcription in vivo. We introduced wild-type and mutant versions of the Bombyx tRNAAla genes into Drosophila Schneider-2 cells and found that the tRNACAla gene is efficiently transcribed and that its transcription depends strongly on the distal segment of its upstream promoter. In contrast, the tRNASGAla gene is inefficiently transcribed, and this inefficiency results from lack of a specific sequence within the distal tRNACAla upstream promoter. This sequence, 5'-TTTATAT-3', is sufficient to increase the activity of the tRNASGAla promoter to that of the tRNACAla promoter. Moreover, promoters containing the 5'-TTTATAT-3' element are stimulated by increased levels of cellular TATA-binding protein. Together these results indicate that, in insect cells, a TATA-like element is specifically required to form functional TATA-binding protein-containing complexes that promote efficient transcription of tRNA genes.

Animals↗
PubMed2003

Inhibition of transfer messenger RNA aminoacylation and trans-translation by aminoglycoside antibiotics.

Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation.

Acylation↗
PubMed1998

Secondary structure dimorphism and interconversion between hairpin and duplex form of oligoribonucleotides.

RNA hairpins can alternatively form a dimer with a bulged loop flanked by regularly base paired regions. [1H]NMR spectroscopy and native gel electrophoresis were used to study how the sequence of nucleotides in the loop of the hairpin affect the hairpin-duplex interconversion. As a model system, a hairpin containing 7 nucleotides in the loop and 5 base pairs in the stem was used. The loop size was gradually reduced from 7 to 4 nucleotides, yielding finally the stable UNCG tetraloop. Single nucleotide mutations were performed to investigate the influence of the self-complementarity of the loop sequence on the dimerization. The results demonstrate that (1) the initial fraction of hairpin is determined by concentration of the oligonucleotide, the annealing procedure, and the relative stability of the loop, (2) the degree of self-complementarity of the loop sequence of the hairpin governs the dimerization kinetics, and (3) oligonucleotides complementary to the loop sequence decrease the dimerization rate. We propose a secondary structure-based model for the dimerization reaction of RNA hairpins in which the formation of intermolecular base pairs between self-complementary nucleotides of the loops represents the nucleation step.

Base Sequence↗
PubMed1998

Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains.

RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.

Alanine-tRNA Ligase↗
PubMed1990

Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2.

An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site. Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor. An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition. An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination. The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified. RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found. If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted. These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon.

Base Sequence↗