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PubMed1983

Coil leads to helix transition in polyadenylic acid induced by the binding of epinephrine, norepinephrine, and isoproterenol: circular dichroism study.

A circular dichroism spectropolarimetric study on the conformation of polyadenylic acid (poly A) in neutral solutions demonstrated a coil leads to helix transition induced by intercalative binding of critical amounts of epinephrine, norepinephrine, and isoproterenol relative to poly A. Theoretical treatment of the experimental data indicated a first-order kinetic transition in poly A. It was possible to measure transition rate constants of the epinephrine-poly A and norepinephrine-poly A systems and to calculate the activation energies. The results indicate a high level of temperature dependence of the rate constants. The effects can be reversed by increasing ionic strength, indicating the significance of the electrostatic interactions. The importance of the results is discussed in terms of the possible role of the catecholamines as control mechanisms for the poly A-regulated translation of the genetic code on mRNA.

Circular Dichroism↗
PubMed2002

Stop codon decoding in Candida albicans: from non-standard back to standard.

The human pathogen Candida albicans translates the standard leucine-CUG codon as serine. This genetic code change is mediated by a novel ser-tRNA(CAG), which induces aberrant mRNA decoding in vitro, resulting in retardation of the electrophoretic mobility of the polypeptides synthesized in its presence. These non-standard decoding events have been attributed to readthrough of the UAG and UGA stop codons encoded by the Brome Mosaic Virus RNA 4, which codes for the virion coat protein, and the rabbit globin mRNAs, respectively. In order to fully elucidate the behaviour of the C. albicans ser-tRNA(CAG) towards stop codons, we have used other cell-free translation systems and reporter genes. However, the reporter systems used encode several CUG codons, making it impossible to distinguish whether the slow migration of the polypeptides is caused by the replacement of leucines by serines at the CUG codons, readthrough, or a combination of both. Therefore, we have constructed new reporter systems lacking CUG codons and have used them to demonstrate that aberrant mRNA decoding in vitro is not a result from stop codon readthrough or any other non-standard translational event. Our data show that a single leucine to serine replacement at only one of the four CUG codons encoded by the BMV RNA-4 gene is responsible for the aberrant migration of the BMV coat protein on SDS-PAGE, suggesting that this amino acid substitution (ser for leu) significantly alters the structure of the virion coat protein. The data therefore show that the only aberrant event mediated by the ser-tRNA(CAG) is decoding of the leu-CUG codon as serine.

Amino Acid Substitution↗
PubMed1997

A rapid fluorescence bioassay for the determination of selenium on agar plates.

The essential trace element selenium (Se) is involved in the form of selenocysteine at the active site of several prokaryotic and eukaryotic proteins called selenoproteins. These proteins have recently attracted attention particularly in relation to their application to human health and new characteristics of the genetic code. We have recently described a selenium bioassay based on a recombinant DNA construct in which the expression of the lac' Z gene in Escherichia coli is proportionally and specifically driven by UGA-directed selenocysteine incorporation. Here we have further developed this bioassay for more rapid and sensitive detection and measurement of selenium that permits screening of the selenium status on agar plates. Again, the inclusion of selenium into the lac'Z-fusion product is reflected by the level of beta-galactosidase activity, which in turn is reflected by the intensity of fluorescence on agar plates. This fluorescing agent is a 4-methylumbelliferyl moiety which is released through the cleavage by the enzyme of 4-methylumbelliferyl-beta-D-galactoside. The intensity of the fluorescence is easily detected by uv irradiation and photographed by polaroid or video cameras.

Agar↗
PubMed1994

Association of deletion and homoplasmic point mutation of the mitochondrial DNA in an ocular myopathy.

The mitochondrial DNA of a 41 year old patient with ocular myopathy was explored. We found a deletion of 3540 base pair in about 50% of the mitochondrial genomes associated with a homoplasmic point mutation. The mutation at nucleotide pair 7444 converts stop codon AGA into lysine codon AAA (human mitochondrial genetic code). The synergistic effect between two point mutations has already been described in mitochondrial pathology but this is the first time that an association between a deletion and a point mutation is shown.

Adult↗
PubMed1998

Development and control of the circadian pacemaker for melatonin release in the chicken pineal gland.

UNLABELLED: Melatonin (MT) release from explanted pineal glands of 3- to 20-week-old chicken was investigated in a 5-day perifusion system. Both the chicken and the explanted glands were exposed to various environmental lighting regimens. OBSERVATIONS: (1) The explanted chicken pineal is sensitive to direct light. Continuous illumination during the in vitro period abolishes the circadian rhythm of the MT secretion in 3 days. Continuous darkness has limited effect. (2) Reverse illumination completely reverses the MT cycle in 2 days. (3) Rhythmic illumination with short (6-h) periods only slightly modulates the MT release pattern: the basic, 24-h periodicity is preserved. (4) The circadian MT pacemaker develops normally and becomes synchronized to the day even if the chicken has never experienced alteration in the environmental illumination (those hatched and bred under continuous illumination). The explanted pineal from these chickens exhibits normal MT cycle and light sensitivity. Conclusion, Chicken pineal contains a complete, genetically coded circadian pacemaker with a fixed frequency. The pacemaker is synchronized to the day by the altered environmental illumination and by at least one other, unknown environmental factor. With altered illumination, in vitro, the 24-h periodicity of the pacemaker cannot be changed significantly, but its phase can be shifted. In contrast to conclusions obtained from in vivo observation in mammals, light seems to stimulate MT secretion from the avian pineal in vitro. For development and daily synchronization of the circadian MT pacemaker in the chicken pineal gland, periodic changes in the environmental illumination are not necessary.

Animals↗
PubMed1998

Expression of p16INK4 and retinoblastoma protein Rb in vulvar lesions of Chinese women.

The protein products of the two tumor suppressor genes located on 9p and 13p, p16INK4 and Rb, respectively, play an important role in regulation of the cell cycle and are implicated in tumorigenesis. We examined 49 cases of benign vulvar lesions, vulvar intraepithelial neoplasia (VIN), and squamous cell carcinoma with immunohistochemical staining to determine expression of p16INK4 and Rb. All and 86% of benign lesions expressed Rb and p16INK4, respectively; 40% each of VIN I and VIN III expressed p16INK4 and Rb, respectively; and 37 and 68% of squamous cell carcinomas expressed p16INK4 and Rb, respectively. The combination of the lack of p16INK4 and/or Rb expression increased from benign lesions (14.3%), through VIN I (60%) and VIN III (60%), to invasive squamous cell carcinoma (72%), thus supporting the postulation that alterations in p16INK4 or Rb could be significant events in progression of disease. The loss of Rb expression also increased from stage I carcinoma (16.7%) through stage II (26.7%) and III (44.4%), to IV (50%), suggesting that Rb may play an important role in tumor progression. A larger study on VIN lesions and genetic coding is suggested to further investigate the role of p16INK4, Rb, and other factors in tumorigenesis and progression of vulvar cancers.

Carcinoma, Squamous Cell↗
PubMed1994

Antibodies to complementary peptides as probes for receptors.

Peptide hormones initiate their physiological responses by binding to receptor proteins embedded in the plasma membranes of their target cells. Mechanisms accounting for specific protein-protein interactions, such as peptide hormone binding by cell receptors or epitope binding by antibody have not been defined. A fundamental tenet of the immunological network hypothesis is the generation of anti-idiotypic antibodies to epitopes located in the hypervariable regions of antibody evoked in the same animal species. Anti-idiotypic antibodies to antibodies to peptide hormones with specificity for epitopes involving antibody binding sites may mimic the actions of the peptide hormone by binding to receptors and evoke cell responses associated with the hormone. A provocative relationship was identified in the genetic code, which recognized that complementary codons for strongly hydrophobic amino acids code for strongly hydrophilic amino acids. This led to the proposal and then to demonstration that peptide pairs based on the nucleotide sequences of complementary codons bind one another. It was then proposed that immunization with complementary peptides to peptide hormones may produce antibodies which, analogous to anti-idiotypic antibodies, may mimic the hormone. Some antibodies to complementary peptides for peptide hormones have been shown to mimic the peptide hormones by binding to their receptors and evoking cell responses characteristic of those of the hormones. Exploiting these relationships, some antibodies to complementary peptides for peptide hormones have been used to identify, purify, and characterize receptor proteins for peptide hormones. Polypeptide hormones initiate their characteristic physiologic effects by binding to specific receptor proteins located on the plasma membranes of their target cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library.

A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a "one bead, one peptide" combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P2, P1 and P'2) with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated alpha-chymotrypsin under appropriate conditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with Ki values in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed.

Amino Acid Sequence↗
PubMed1996

RNA sequence determinants for aminoglycoside binding to an A-site rRNA model oligonucleotide.

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucleotide that mimics the A site of Escherichia coli 16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The antibiotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407.G1494 base-pair, and A.U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.

Aminoglycosides↗
PubMed1998

Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA.

Aminoglycoside antibiotics that bind to ribosomal RNA in the aminoacyl-tRNA site (A-site) cause misreading of the genetic code and inhibit translocation. An A-site RNA oligonucleotide specifically binds to aminoglycoside antibiotics and the structure of the RNA-paromomycin complex was previously determined by nuclear magnetic resonance (NMR) spectroscopy. Here, the A-site RNA structure in its free form has been determined using heteronuclear NMR and compared to the structure of the paromomycin-RNA complex. As in the complex with paromomycin, the asymmetric internal loop is closed by a Watson-Crick base-pair (C1407.G1494) and by two non-canonical base-pairs (U1406.U1495, A1408.A1493). A1492 stacks below A1493 and is intercalated between the upper and lower stems. The comparison of the free and bound conformations of the RNA shows that two universally conserved residues of the A site of 16 S rRNA, A1492 and A1493, are displaced towards the minor groove of the RNA helix in presence of antibiotic. These changes in the RNA conformation place the N1 positions of A1492 and A1493 on the minor groove side of the A-site RNA and suggest a mechanism of action of aminoglycosides on translation.

Anti-Bacterial Agents↗
PubMed1998

Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16 S rRNA.

Aminoglycoside antibiotics that bind to ribosomal RNA in the aminoacyl-tRNA site (A-site) cause misreading of the genetic code and inhibit translocation. We have recently solved the structure of an A-site RNA-paromomycin complex. The structure suggested that rings I and II, common to all aminoglycosides that bind to the A-site, are the minimum motif for specific ribosome binding to affect translation. This hypothesis was tested biochemically and with a detailed comparative NMR study of interaction of the aminoglycosides paromomycin, neomycin, ribostamycin, and neamine with the A-site RNA. Our NMR data show that rings I and II of neomycin-class aminoglycosides are sufficient to confer specificity to the binding of the antibiotics to the model A-site RNA. Neomycin, paromomycin, ribostamycin and neamine bind in the major groove of the A-site RNA in a unique binding pocket formed by non-canonical base pairs and a bulged nucleotide. Similar NMR properties of the RNA and the diverse antibiotics within the different complexes formed with neomycin, paromomycin, ribostamycin and neamine suggest similar structures for these complexes.

Anti-Bacterial Agents↗
PubMed1998

Antibiotic inhibition of RNA catalysis: neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions.

The aminoglycoside antibiotic neomycin B induces misreading of the genetic code during translation and inhibits several ribozymes. The self-splicing group I intron derived from the T4 phage thymidylate synthase (td) gene is one of these. Here we report how neomycin B binds to the intron RNA inhibiting splicing in vitro. Footprinting experiments identified two major regions of protection by neomycin B: one in the internal loop between the stems P4 and P5 and the other in the catalytic core close to the G-binding site. Mutational analyses defined the latter as the inhibitory site. Splicing inhibition is strongly dependent on pH and Mg2+ concentration, suggesting electrostatic interactions and competition with divalent metal ions. Fe2+-induced hydroxyl radical (Fe-OH.) cleavage of the RNA backbone was used to monitor neomycin-mediated changes in the proximity of the metal ions. Neomycin B protected several positions in the catalytic core from Fe-OH. cleavage, suggesting that metal ions are displaced in the presence of the antibiotic. Mutation of the bulged nucleotide in the P7 stem, a position which is strongly protected by neomycin B from Fe-OH. cleavage and which has been proposed to be involved in binding an essential metal ion, renders splicing resistant to neomycin. These results allowed the docking of neomycin to the core of the group I intron in the 3D model.

Anti-Bacterial Agents↗
PubMed1999

Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA.

Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes. The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule. Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance. Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix. This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region. Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation. Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced. We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed. The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix.

Acylation↗
PubMed1999

Stitching together RNA tertiary architectures.

The powerful explanatory paradigm of molecular biology requiring form to co-evolve with function has again been proven successful when, over the recent two decades, a wealth of biological functions have been uncovered for RNA. Previously considered as a mere mediator of the genetic code, RNA is now acknowledged as a key player in a wide variety of cellular processes. Along with the discovery of novel biological functions of RNA molecules, a number of RNA three-dimensional structures have been solved which beautifully demonstrate the molecular adaptability which allows RNA to participate as a key player in these functions. A distinct repertoire of molecular motifs provides a basis for the assembly of complex RNA tertiary architectures.

Base Pair Mismatch↗
PubMed2001

Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase.

Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code. Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vivo. In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations. Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort. Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities. The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design. The binding of Asn in an alternate, "head-to-tail" orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the "Asp-like" orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase. It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The double mutation (K198L,D233E) has a similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the "Asp-like" binding affinities calculated here. In molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1-2 A compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis.

Amino Acid Substitution↗
PubMed2001

Construction of an Escherichia coli knockout strain for functional analysis of tRNA(Asp).

The specific aminoacylation of tRNA is critical for translation of the genetic code. A molecular description of aminoacylation requires knowledge of the relevant three-dimensional structures, biochemical parameters and the structure-function relationship of the synthetase and its substrate tRNA. Extensive structural and biochemical data are available on the aspartic acid system of Escherichia coli, but there is a paucity of cellular functional data. We have developed a system to overcome this deficiency by engineering an E. coli knockout tRNA(Asp) strain, thereby allowing a penetrating analysis of tRNA(Asp) structure and function under conditions that prevail in the cell.

Acylation↗
PubMed1996

Organization of the mouse cardiac natriuretic peptide locus encoding BNP and ANP.

The genes encoding the mouse atrial natriuretic peptide and B-type natriuretic peptide were previously shown to be physically linked on mouse chromosome 4 (Steinhelper ME, 1993, Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene. Circ Res 72: 984-992). In the present study the spatial relationship and orientation of the mouse atrial natriuretic peptide and B-type natriuretic peptide transcription units were identified and a physical map of the mouse cardiac natriuretic peptide locus was obtained. To this end, genomic clones encoding atrial natriuretic peptide and B-type natriuretic peptide were isolated from a mouse genomic library in bacteriophage P1. Three independent clones encoding atrial natriuretic peptide were isolated and two of these also encode B-type natriuretic peptide. Both transcripts were shown to arise from the same DNA strand, with B-type natriuretic peptide encoded approximately 15 kb 5'-of atrial natriuretic peptide based on field inversion gel electrophoresis of fragments amplified with specific oligonucleotides. This finding was confirmed by isolation of subclones comprising the entire locus and by blot hybridization analysis of mouse genomic DNA. The results show that the genes encoding the two natriuretic peptides expressed predominantly in mammalian cardiac myocytes are organized in tandem on mouse chromosome 4. This information provides a physical framework for investigating mechanisms that regulate transcription of the cardiac natriuretic peptide locus.

Animals↗