Part of the matrix on the 5' side from codon in the E-segment is close to protein S26 on the human 80S ribosome

Positioning of mRNA on the 80S ribosome upstream the E site bound codon was studied using derivatives of nona- and dodecaribonucleotides containing the triplet UUU coding for Phe at the 3'-terminus, and a perfluorophenylazide cross-linker on either the first or the third nucleotide. Two sets of the mRNA analogues were used, with the photoactivatable groups on either the C5 atom of the uridine or the N7 atom of the guanosine. The modified nucleotides were directed to positions from -4 to -9 with respect to the first nucleotide of the P site bound codon by tRNA(Phe) cognate to the triplet UUU targeted to the P site. Mild UV-irradiation of ribosomecomplexes with tRNA(Phe) and mRNA analogues resulted in the cross-linking to the 40S subunits preferentially, mainly to the proteins. The principal target for the cross-linking was protein S26 in all cases. Location of the photoactivatable group on the nucleotide at position -4 lead also to the minor cross-linking to protein S3, and at position -6 to protein S14. In the absence of tRNA, all mRNA analogues cross-linked to protein S3.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.     

Interaction of T. thermophilus phenylalanyl-tRNA synthetase with the 3'-terminal nucleotide of tRNAPhe

The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value. Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76. The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76. The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS. The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.     

Photochemical cross-linking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop

The 2-thiocytidine residue at position 32 of tRNA1Arg from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetylarginyl-tRNA1Arg derivatives were cross-linked to the P site of E. coli 70S ribosomes by irradiation. Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength. From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin. Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases. Two of the N-acetylarginyl-tRNA1Arg derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA. Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule. In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.