How glutaminyl-tRNA synthetase selects glutamine.

BACKGROUND: Aminoacyl-tRNA synthetases covalently link a specific amino acid to the correct tRNA. The fidelity of this reaction is essential for accurate protein synthesis. Each synthetase has a specific molecular mechanism to distinguish the correct pair of substrates from the pool of amino acids and isologous tRNA molecules. In the case of glutaminyl-tRNA synthetase (GlnRS) the prior binding of tRNA is required for activation of glutamine by ATP. A complete understanding of amino acid specificity in GlnRS requires the determination of the structure of the synthetase with both tRNA and substrates bound. RESULTS: A stable glutaminly-adenylate analog, which inhibits GlnRS with a Ki of 1.32 microM, was synthesized and cocrystallized with GlnRS and tRNA2Gln. The crystal structure of this ternary complex has been refined at 2.4 A resolution and shows the interactions made between glutamine and its binding site. CONCLUSIONS: To select against glutamic acid or glutamate, both hydrogen atoms of the nitrogen of the glutamine sidechain are recognized. The hydroxyl group of Tyr211 and a water molecule are responsible for this recognition; both are obligate hydrogen-bond acceptors due to a network of interacting sidechains and water molecules. The prior binding of tRNAGln that is required for amino acid activation may result from the terminal nucleotide, A76, packing against and orienting Tyr211, which forms part of the amino acid binding site.

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro.

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases.

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.

Chemical nucleic acid synthesis, modification and labelling.

The chemical synthesis of RNA on solid phase has now become routine using labile protecting groups and mild deprotection methods. The great interest in antisense technology has sparked the development of P-chiral phosphorothioates and a large number of DNA analogues with modified sugars and/or backbones to increase resistance to nucleases, and with modifier groups attached to the sugar, nucleobase or internucleotide function to aid cellular uptake.

Antisense oligonucleotides made of 2'-O-alkylRNA: their properties and applications in RNA biochemistry.

Oligo(2'-O-alkylribonucleotides) have been developed recently as novel oligonucleotide analogues with properties that enhance their use as antisense probes. They possess high chemical stability and are resistant to hydrolysis by DNA- or RNA-specific nucleases. Many forms of oligo(2'-O-alkylribonucleotides) hybridise specifically and efficiently to complementary RNA sequences, forming stable duplexes that are not substrates for cleavage by RNAse H. In combination with prosthetic reporter groups, such as biotin, DNP or fluorophores, oligo(2'-O-alkylribonucleotides) have important applications in a wide range of biochemical studies on RNA function and structure.

Comparative evaluation of seven oligonucleotide analogues as potential antisense agents.

12-Mer analogues, representative of seven different classes of structurally modified oligonucleotides and complementary to the same target, have been compared for their binding affinity for both single-stranded DNA and RNA, resistance to hydrolysis by nucleases in culture medium (RPMI 1640 + 10% inactivated fetal calf serum), and inhibition of HIV-1 replication in de novo infected MT4 T lymphocytes. The viral target was the splice acceptor site of the premessenger coding for the regulatory protein tat. The oligo(2'-O-alkyl)ribonucleotides (beta-2'O-allyl-RNA and beta-2'OMe-RNA) were shown to form the most stable hybrids with complementary RNA strands whereas the alpha-anomeric oligodeoxynucleoside phosphorothioate analogue displayed the highest stability in the culture medium. All the modified oligonucleotides examined in the present study exhibited a sequence-nonspecific inhibitory effect on HIV-1 replication, the phosphorothioate analogues being the most active ones (ED50 < 1 microM).

Dopamine D(2) receptor ribozyme inhibits quinpirole-induced stereotypy in rats.

The injection of a dopamine D(2) receptor hammerhead ribozyme (20 microg) once daily for 5 days into the nucleus accumbens of rats resulted in an inhibition of stereotyped sniffing and locomotor activation produced by the selective dopamine D(2) receptor agonist, quinpirole (0.4 mg kg(-1) s.c.). The results suggest that ribozymes may be useful in the study of in vivo gene function in the brain.

Chemistry and applications of oligonucleotide analogues.

This review is aimed at biochemists and molecular biologists, and covers the chemistry and key features involved in the solid-phase synthesis of a variety of the better known DNA and RNA analogues by the phosphoramidite and H-phosphonate methods. A wide spectrum of biological applications such as inhibition of gene expression, translation arrest, RNA processing, affinity purification of RNA-protein complexes, in situ hybridization, and synthetic ribozymes are then discussed in some detail, enabling the molecular biologist to get an idea of what is possible using the current technology.

A non-radioactive automated method for DNA sequence determination.

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.

A new linkage for solid phase synthesis of oligodeoxyribonucleotides.

An aryl diisocyanate has been used to attach an appropriately protected 2'-deoxyribonucleoside bearing a free 3'-hydroxyl group, to a long chain alkylamine controlled pore glass support via a urethane moiety, in a simple two step procedure. This obviates the need for the preparation and short column chromatographic purification of the 2'-deoxyribonucleoside-3'-O-succinates required for preparation of the widely used succinyl linked supports. The greater stability of the urethane bond compared to an ester bond led to substantially higher yields of oligodeoxyribonucleotides prepared by the solid phase phosphotriester method. More than twenty oligodeoxyribonucleotides have already been synthesized on the glass support bearing the new linkage.

Chemical synthesis of a gene for somatomedin C.

A synthetic gene for somatomedin C, a human growth factor, has been assembled by a single ligation of 23 oligodeoxyribonucleotides, which were chemically synthesized by an improved solid phase phosphotriester method.

Evidence for an associative mechanism in the phosphoryl transfer step catalyzed by rabbit muscle creatine kinase.

Creatine kinase does not catalyze the scrambling of 18O in adenosine 5'-[alpha beta-18O, beta-18O2]triphosphate in the absence of creatine, in the presence of L-arginine or taurocyamine (competitive inhibitors of creatine), or in the presence of poor substrates where single turnover experiments were performed. In order to support this prima facie evidence for an associative mechanism of phosphoryl transfer, an investigation was undertaken of 1-carboxymethyl-2-aminoimidazole, a new substrate analogue of creatine. This analogue has a binding constant for rabbit muscle creatine kinase similar to creatine and 1-carboxymethyl-2-iminoimidazolidine, but the initial rate of phoshorylation by MgATP in the presence of creatine kinase is almost 5 orders of magnitude slower. The phosphorylation product, assigned the structure 1-carboxymethyl-2-imino-3-phospho-4-imidazoline is also a poor substrate for the phosphorylation of MgADP by creatine kinase. These observations can be accounted for by an associative SN2(P) mechanism of phosphoryl transfer and by a microenvironment of the enzyme-bound creatine (or creatine analogue) which lowers the pKa of the guanidino group by several pH units compared with that in aqueous solution.

Sequence-specific affinity selection of mammalian splicing complexes.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/biotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.

New synthetic routes to protected purine 2'-O-methylriboside-3'-O-phosphoramidites using a novel alkylation procedure.

A highly selective alkylation procedure has been developed enabling new synthetic routes to be established for protected purine 2'-O-methylriboside-3'-O-phosphoramidites; building blocks for the assembly of 2'-O-methyloligoribonucleotides. The new procedure avoids the use of the highly toxic and potentially explosive reagent diazomethane and is far superior to the use of silver oxide/methyl iodide. Moreover, the use of highly versatile key intermediates will enable the synthesis of a wide variety of base modified analogues as well as other 2'-O-alkylriboside derivatives.

The synthesis of protected 5'-mercapto-2',5'-dideoxyribonucleoside-3'-O-phosphoramidites; uses of 5'-mercapto-oligodeoxyribonucleotides.

The syntheses of the four novel, base protected 5'-(S-triphenylmethyl)mercapto-2',5'-dideoxyribonucleoside-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) are described. These compounds have been used to prepare 5'-(S-triphenylmethyl) mercapto-oligodeoxyribonucleotides, which are readily purified by reversed phase h.p.l.c., owing to the highly lipophilic trityl group. After cleavage of the S-trityl group by silver or mercuric ions, the free thiol moiety can be coupled to a wide variety of reagents, generating very useful probes. Fluorescent labelled 5'-mercapto-oligodeoxyribonucleotides are being used for automated DNA sequencing without radioactivity, and heavy metal labelled 5'-mercapto-oligonucleotides will be used in X-ray crystallography.

Highly efficient oligodeoxyribonucleotide synthesis using fully base protected phosphodiester building blocks carrying 2-(1-methylimidazol-2-yl) phenyl protection of the phosphate.

Four fully base protected phosphodiester building blocks have been synthesised and fully characterised. The phosphate protecting group used was the 2-(1-methylimidazol-2-yl)phenyl group, enabling intramolecular catalysis of the condensation step in oligodeoxyribonucleotide synthesis by the solid phase phosphotriester method. Cycle times of about 12 min could thus be achieved. Moreover, the used of extra protecting groups on deoxythymidine and 2'-deoxyguanosine resulted in much cleaner oligodeoxyribonucleotides as evidenced by ion-exchange and reversed phase h.p.l.c.

The synthesis of protected 5'-amino-2',5'-dideoxyribonucleoside-3'-O-phosphoramidites; applications of 5'-amino-oligodeoxyribonucleotides.

Synthetic routes to the four appropriately protected 5'-amino-2',5'-dideoxyribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) have been developed. The structures of all intermediates were confirmed by 13C n.m.r. spectroscopy. These building blocks have been used to prepare 5'-amino-oligodeoxyribonucleotides, which can be coupled to a wide variety of compounds, in particular metal cluster derivatives, but also fluorophores and biotin derivatives, thus generating a variety of very useful probes. Brief mention is made of a tetrairidium cluster derivative of 5'-amino-d[CCGATATCGG], which has been cocrystallised with EcoRV, and will be used for electron microscopy studies.

Nuclease resistant ribozymes with high catalytic activity.

Hammerhead ribozymes are efficient RNA enzymes characterized by a typical hammerhead secondary structure and a number of conserved bases. Little is known about the role of the ribose-phosphate backbone, although it is obviously important since a DNA molecule with the same base sequence is not a catalyst. Here we describe the synthesis of artificial ribozymes where modified (2'-O-allyl- and 2'-O-methyl-) ribonucleotides substitute for the corresponding ribonucleotides. A systematic analysis of partially substituted polymers identified a minimum set of six non-contiguous positions where insertion of modified ribonucleotides strongly affects catalytic activity. Surprisingly, ribozymes completely substituted except for these six ribonucleotides are still very active. These molecules efficiently cleave in trans target RNAs in a sequence-specific way, but, unlike RNA ribozymes, are very resistant to nuclease degradation and are very stable in serum. These properties make such synthetic polymers potentially useful for in vivo gene expression studies and therapeutic applications.

Preparation of a novel psoralen containing deoxyadenosine building block for the facile solid phase synthesis of psoralen-modified oligonucleotides for a sequence specific crosslink to a given target sequence.

4,5',8-Trimethylpsoralen was attached to the C8-position of deoxyadenosine via a sulfur atom and a five carbon atom linker. The modified deoxyadenosine was then converted to a protected phosphoramidite and used as unusual as a building block for solid phase oligodeoxyribonucleotide synthesis. The efficiency of the photoreaction of a psoralen-modified oligonucleotide to a complementary matrix strand reached more than 90% within a 1 hour irradiation time at a wavelength of 345 nm.