Antibacterial nitroacridine, Nitroakridin 3582: binding to nucleic acids in vitro and effects on selected cell-free model systems of macromolecular biosynthesis

Nitroakridin 3582 (NA) formed complexes with native deoxyribonucleic acid (DNA) and with transfer ribonucleic acid (tRNA) species from Escherichia coli. Spectrophotometric titrations of NA with these nucleic acids produced numerical results from which nonlinear adsorption isotherms were derived. These curves indicated the existence of more than one class of binding sites on the polymers to which NA was bound by more than one process. The stoichiometry of strong binding of NA to double helical DNA was in agreement with a conventional value (1 ligand molecule per 4.2 component nucleotides) for complete intercalation binding. NA inhibited the DNA-dependent DNA polymerase I and RNA polymerase reactions, the first strongly and the second appreciably. These inhibitions corresponded to the extents to which NA inhibits DNA and RNA biosyntheses in vivo. Evidently, NA interferes with the template function of DNA. The drug also inhibited the polymerization of phenylalanine in a cell-free E. coli ribosome-polyuridylic acid [poly (U)] system. The effect paralleled an inhibition of the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. Ethidium bromide acted similarly. The antimalarial drug, chloroquine, stimulated polyphenylalanine synthesis, apparently as a result of stimulating the poly (U)-directed binding of phenylalanyl tRNA to ribosomes.     

Prokaryotic and eukaryotic tetrameric phenylalanyl-tRNA synthetases display conservation of the binding mode of the tRNA(Phe) CCA end

The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.     

Biochemical research on oogenesis. Binding of tRNA to the nucleoprotein particles of Xenopus laevis previtellogenic oocytes

In previtellogenic oocytes of Xenopus laevis, nearly all tRNA is included in nucleoprotein particles (thesaurisomes) sedimenting at 42 S. We evaluate the possibility of a tRNA exchange between the particles and the ribosomes during protein synthesis. We find that the particles take up tRNA after a very short incubation in vitro. In the absence of ATP, the particles preferentially bind charged tRNA. In the presence of ATP, more tRNA binds to the particles, and the sedimentation coefficient of the integrated tRNA is displaced to 45 S. When added to nonfractionated homogenates of oocytes together with ATP, poly(U) strongly stimulates the incorporation of radioactive phenylalanine into tRNA and protein. The labeled protein (polyphenylalanine) cosediments with the ribosomes, whereas most of phenylalanyl tRNA cosediments with the thesaurisomes. These data suggest that the thesaurisomes participate to some extent in protein synthesis. They release charged tRNA, thereby supplying the ribosomes with activated amino acids. Discharged tRNA is then taken up, reacylated, and stored in the particles until the next round of peptide bond formation. The aminoacylation and storage functions are probably carried out by two very unequal populations of particles. The main subclass of particles (42 S) binds and stores tRNA in an ATP-independent manner. A much smaller subclass of particles (45 S) is responsible for reacylation of discharged tRNA.     

Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA

A set of 45 different tRNAs, each containing a single deoxynucleotide substitution covering the upper half of the molecule was used in conjunction with a high-throughput ribonuclease protection assay to investigate the thermodynamic role of 2' hydroxyl groups in stabilizing a complex with elongation factor Tu (EF-Tu) from Thermus thermophilus. Five distinct 2' hydroxyl groups were identified where substitution with a proton resulted in an approximately tenfold decrease in the binding affinity. The same five 2' hydroxyl groups reduced the affinity of the interaction with the nearly identical Thermus aquaticus EF-Tu. Four of these 2' hydroxyl groups were observed to form hydrogen bonds in a co-crystal structure of tRNA(Phe) and T. aquaticus EF-Tu, while the fifth 2' hydroxyl group can be associated with an intramolecular hydrogen bond in the tRNA. However, four additional hydrogen bonds to 2' hydroxyl groups observed in the crystal structure show no thermodynamic effect upon disruption. Some of these discrepancies may be reconciled based on the unbound structures of the protein and RNA.     

Chemical aminoacylation of transfer ribonucleic acid. The reaction of Escherichia coli tRNA with ethyl N-benzyloxycarbonylorthoglycinate.

The alpha-carbethoxypentadecyltrimethylammonium (Septonex) salt of tRNA (Ib) was condensed with ethyl N-benzyloxycarbonylorthoglycinate (II) in dimethylformamide in vacuo and in the presence of H3PO4 as catalyst. Pancreatic RNAase degradation and phenylalanine acceptor activity showed a 55--60% conversion to the 2',3'-cyclic orthoglycinate derivative of tRNA (IIIb). The orthoester grouping of IIIb was quantitatively hydrolyzed in 80% formic acid at 0 degrees C for 15 min to give 2'(3')-O-(N-benzyloxycarbonyl)glycyl tRNA (IVb). The latter was stripped at pH 8.8 to give tRNA whose behavior on DEAE cellulose column and gel electrophoresis was similar to that of starting tRNA. The phenylalanine acceptor activity amounted to almost 80% of the starting tRNA.     

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities ofEscherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg²⁺ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg²⁺ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.     

Phenylalanyl synthetase function in cultured fibroblasts from subjects with progeria.

Since progeria cells contain a diversity of altered proteins, some aspects of phenylalanyl synthetase function were examined in semipurified extracts of cultured skin fibroblasts using mixed rabbit tRNA as acceptor. No significant differences were found in the Km and Vmax for phenylalanine or ATP in progeria cells compared with controls. Initial velocities of both progeria and control synthetases were lower at late passage owing to either reduced enzyme content or reduced catalytic efficiency. Reverse phase 5 chromatography of tRNAs acylated by progeria and control synthetases gave a single peak of labeled phenylalanine tRNA in all cases with no secondary peaks evident. Total activity of phenylalanyl synthetase in progeria cells was similar to that of control cells at early passage while late-passage control cells had lower specific activities of these synthetases per unit protein.     

Testing the classical two-tRNA-site model for the ribosomal elongation cycle

An experimental system where the elongation of a polypeptide (polyphenylalanine) is performed stepwise and synchronously by purified Escherichia coli ribosome in a matrix-coupled poly (U) column is proposed for testing the number of non-overlapping tRNA binding sites on the elongating ribosome. If phenylalanyl[3H]tRNA is introduced into the column and bound with the ribosomes at the beginning of a given elongation cycle, deacylated [3H]tRNA is shown to be released from the ribosomes and comes out from the column at the translocation step of the next elongation cycle. The result obtained is fully predicted by the classical two-tRNA-site model and contradicts any model involving more than two non-overlapping high-affinity tRNA binding sites in the ribosomal elongation cycle.     

Footprinting mRNA-ribosome complexes with chemical probes.

We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.     

An extracellullar nuclease from Bacillus firmus VKPACU-1: specificity and mode of action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5'UMP > 5'AMP > 5'GMP where as in hydrolysis of DNA the mononucleotides in the order of 5'dAMP > 5'dGMP > 5'dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3' hydroxyl and 5' phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Purification and properties of a ribonuclease in human urine that hydrolyses polycytidylic acid.

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylamide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is acidic and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21,500. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3'-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2': 3'-cyclic phosphate or uridine 2':3'-cyclic phosphate. Its rate of hydrolysis of poly (U) is less than 2% of that of poly C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U). It differs from bovine pancreatic Rnase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Preparation and characterisation of ribonuclease from human hypertrophic prostate gland (RNAase P2).

An endo-type, cyclising, 3'-phosphate-forming rebonuclease was purified to homogeneity from a water/Tween 80 extract of human hypertrophic prostate gland. The enzyme is acid- and heat- resistant and is optimally active at pH 7.0, 0.1 M NaCl. Molecular weight determined by gel filtration on Sephadex G-75 and sucrose density gradient centrifugation gave a mean value of 15 000. The prostatic ribonuclease is inhibited by Cu2+, bromoacetate and photooxidation in the presence of methylene blue. Other divalent ions, EDTA and p-chloromercuribenzoate have no influence on the enzymic activity. Prostatic RNase resembles RNase A in that it preferentially cleaves linkages in RNA after pyrimidine nucleotides to produce oligonucleotides terminated in cyclic 2',3' phosphate. The enzyme is inactive with poly(A) - poly(U) as substrate. Poly(U) is hydrolyzed four times as fast as poly(C), and 1.2 times as fast as RNA.     

Concurrent molecular recognition of the amino acid and tRNA by a ribozyme.

We have recently reported an in vitro-evolved precursor tRNA (pre-tRNA) that is able to catalyze aminoacylation on its own 3'-hydroxyl group. This catalytic pre-tRNA is susceptible to RNase P RNA, generating the 5'-leader ribozyme and mature tRNA. The 5'-leader ribozyme is also capable of aminoacylating the tRNA in trans, thus acting as an aminoacyl-tRNA synthetase-like ribozyme (ARS-like ribozyme). Here we report its structural characterization that reveals the essential catalytic core. The ribozyme consists of three stem-loops connected by two junction regions. The chemical probing analyses show that a U-rich region (U59-U62 in J2a/3 and U67-U68 in L3) of the ribozyme is responsible for the recognition of the phenylalanine substrate. Moreover, a GGU-motif (G70-U72) of the ribozyme, adjacent to the U-rich region, forms base pairs with the tRNA 3' terminus. Our demonstration shows that simple RNA motifs can recognize both the amino acid and tRNA simultaneously, thus aminoacylating the 3' terminus of tRNA in trans.     

Poly(U)-dependent polyphenylalanine and polytyrosine synthesis in vitro by a tRNATyr variant with an enzymatically altered anticodon, G-A-A

A variant of T. utilis tRNATyr containing a base substitution (psi----A) in the middle position of the anticodon has been constructed by enzymatic procedures in vitro. This variant is unique in that it can accept both tyrosine and phenylalanine. This tRNA was shown to be active in transferring both tyrosine and phenylalanine into polypeptides in a cell-free, poly (U)-directed translation system from yeast. This result gives further support to the adapter hypothesis since tyrosine, attached to the variant tRNATyr with an anticodon G-A-A, is incorporated into polypeptides in response to poly (U) message.     

Feedback inhibition of the DAHP synthetases by tRNA in Saccharomyces cerevisiae

Summary The use of tyrosine inhibited and phenylalanine inhibited mutants in yeast makes possible the study of the properties of the two 3 deoxy-D-arabino-heptulosonic-acid-7-phosphate synthetases separately. The measurement of the activity of these two enzymes revealed the presence of endogenous inhibitors of the reaction in the crude extracts. These inhibitors are non dialysable, thermostable and sensitive to an RNAse treatment. The use of purified tRNAs showed that the phenylalanine sensitive enzyme is inhibited by both charged and uncharged phenylalanyl tRNA whereas the tyrosine sensitive enzyme is inhibited only by charged tyrosyl tRNA whereas the tyrosine sensitive enzyme is inhibited only by charged tyrosyl tRNA. Both enzymes are also inhibited by the free amino acids, i.e. respectively tyrosine and phenylalanine.     

Peptide Synthesis by Extracts from Bacillus subtilis Spores

Cell-free peptide synthesis by extracts from vegetative cells and spores of Bacillus subtilis was analyzed and compared. The initial rate of phenylalanine incorporation in a polyuridylate-directed system was found to be in a similar range for the two extracts. However, spore extracts frequently incorporated less total phenylalanine as did the vegetative cell system. Optimal conditions for amino acid incorporation by spore extracts were found to be similar to those of vegetative cell extracts. Polyphenylalanine synthesis was stimulated by preincubation of both extracts prior to the addition of polyuridylic acid (poly U) and labeled phenylalanine. Both systems showed a dependence on an energy-generating system and were inhibited by chloramphenicol and puromycin. Ribonuclease, but not deoxyribonuclease, inhibited the reaction significantly. The presence of methionine transfer ribonucleic acid (tRNA(F)) and methionyl-tRNA(F) transformylase was demonstrated in spore extracts. An analysis of several aminoacyl-tRNAs in spores revealed that the relative amounts of these tRNAs were similar to those found in vegetative cells. Only lysine tRNA was found to be present in relatively greater amounts in spores. These results indicate that dormant spores of B. subtilis contain the machinery for the translation of genetic information.     

Oligoribonucleotides containing 2',5'-phosphodiester linkages exhibit binding selectivity for 3',5'-RNA over 3',5'-ssDNA.

Oligoribonucleotides containing 2',5'-phosphodiester linkages have been synthesized on a solid support by the 'silyl-phosphoramidite' method. The stability of complexes formed between these oligonucleotides and complementary 3',5'-RNA strands have been studied using oligoadenylates and a variety of oligonucleotides of mixed base sequences including phosphorothioate backbones. In many cases, particularly for 2',5'-linked adenylates, the UV melting profiles are quite sharp and exhibit large hyperchromic changes. Substituting a few 3',5'-linkages with the 2',5'-linkage within an oligomer lowers the Tm of the complex and the degree of destabilization depends on the neighboring residues and neighboring linkages. The 2',5'-linked oligoribonucleotides prepared in this study exhibited remarkable selectivity for complementary single stranded RNA over DNA. For example, in 0.01 M phosphate buffer--0.10 M NaCl (pH 7.0), no association was observed between 2',5'-r(CCC UCU CCC UUC U) and its Watson-Crick DNA complement 3',5'-d(AGAAGGGAGAGGG). However, 2',5'-r(CCC UCU CCC UUC U) with its RNA complement 3',5'-r(AGAAGGGAGAGGG) forms a duplex which melts at 40 degrees C. The decamer 2',5'-r(Ap)9A forms a complex with both poly dT and poly rU but the complex [2',5'-r(Ap)9A]:[poly dT] is unstable (Tm, -1 degree C) and is seen only at high salt concentrations. In view of their unnatural character and remarkable selectivity for single stranded RNA, 2',5'-oligo-RNAs and their derivatives may find use as selective inhibitors of viral mRNA translation, and as affinity ligands for the purification of cellular RNA.     

The phenylalanine tRNA from Mycoplasma sp. (Kid): a tRNA lacking hypermodified nucleosides functional in protein synthesis

Phenylalanine tRNA from Mycoplasma sp. (Kid) was purified and characterized. The tRNA can be aminoacylated by phenylalanyl-tRNA synthetase from both Mycoplasma and E. coli. In a tRNA-dependent cell-free E. coli amino acid incorporating system programmed with poly U pure Mycoplasma tRNA(Phe) was fully active in promoting phenylalanine incorporation, even in direct competition with homologous E. coli tRNA(Phe). Since the Mycoplasma tRNA lacks isopentenyladenosine, or any related hypermodified nucleoside, it appears that the presence of such nucleosides in tRNA is not an absolute requirement for protein synthesis.     

Is there proofreading during polypeptide synthesis?

The stoichiometric efficiency with which ternary complexes containing Phe-tRNAphe and Leu-tRNAleu support polypeptide synthesis has been compared in a poly(U)-directed, steady-state translation system. When unfractionated tRNA is used to support synthesis, the number of discharged ternary complexes per peptide bond formed is an average of 48 times greater for leucine than for phenylalanine. When three purified leucine isoacceptor species are tested, they each show a characteristic ratio of ternary complexes discharged per missense insertion, normalized to that for phenylalanine: these are 103, 76, and 45 for Leu- tRNA2leu , Leu- tRNA3leu , and Leu- tRNA4leu , respectively. The data are consistent with the functioning of a proofreading mechanism during translation.     

Phenylalanyl-tRNA synthetase of Escherichia coli K10. Effects of zinc(II) on partial reactions of diadenosine 5',5"'-P1,P4-tetraphosphate synthesis, conformation, and protein aggregation

The synthesis of diadenosine 5',5"'-P1-,P4-tetraphosphate (Ap4A) catalyzed by phenylalanyl-tRNA synthetase in the presence of Zn2+ involves the same partial reactions (synthesis of phenylalanyladenylate and transfer of the adenylate moiety to ATP) as occur in the absence of this metal ion. However, transfer is strongly stimulated while adenylate synthesis is depressed. Also inhibited are pyrophosphorolysis of phenylalanyladenylate and transfer of phenylalanine from the adenylate to cognate tRNA, because overall tRNA phenylalanylation is depressed [Mayaux, J.-F., & Blanquet, S. (1981) Biochemistry 20, 4647-4654], whereas binding of tRNA to the synthetase is not. At moderate concentrations of Zn2+, and in the presence of 5 microM phenylalanine and 0.5 mM ATP, transfer of AMP is rate limiting, while at higher concentrations of Zn2+ synthesis of adenylate is rate determining. The Zn2+ concentration optimum for stimulation depends on the concentration of phenylalanine and ATP. The effects of Zn2+ are mediated through two classes of binding site(s) on the synthetase, the half-saturations of which are 1-4 and 20-30 microM Zn2+, respectively. Binding of Zn2+ to the second class of site(s) causes inhibition of the synthetase, whereas binding to the first class is responsible for activation and inhibition, which may be caused by a conformational change. Evidence for the latter is the observed decrease in protein intrinsic fluorescence intensity and the decrease in fluorescence intensity of 6-(p-toluidinyl)naphthalene-2-sulfonate, which is used as a reporter group. The kinetics of the binding reaction show a saturation dependence on Zn2+, also suggesting that a conformational change occurs.(ABSTRACT TRUNCATED AT 250 WORDS)