Interaction of tRNA with ribosomes

Definition of the site of tRNA-binding to ribosomes is suggested on the basis of a free energy of tRNA-ribosome interaction. From this point of view disagreements that have arisen in recent years concerning the numbers of tRNA binding sites on the ribosome, their distribution between subunits, the properties of the third site E in ribosomes and the compatibility of new experimental data with different models of elongation cycle are discussed. The observation of the third site in the ribosome (messenger independent and with a presumably exit function) is not a refutation but an extension of Watson's model of translating ribosome.     

Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.

Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.     

Interaction of cytochrome c with tRNA and other polynucleotides

Interaction between tRNA and other polynucleotides with cytochrome c was studied by visible spectroscopy, fluorescence spectroscopy and gel mobility shift assay in view of the recently reported important regulatory role of tRNA in cytochrome c mediated apoptotic pathway. Visible spectroscopy showed perturbation in the heme binding environment in cytochrome c with tRNA binding. Fluorescence titrations indicated that cytochrome c binds to different polynucleotides with differing affinities. A weak binding was observed with single stranded polyribonucleotides and polydeoxyribonuleotides and strong binding with tRNA and double stranded DNA as indicated by extent of fluorescence quenching and binding constants. Calculation of thermodynamic binding parameters from fluorescence titrations indicated that three molecules of cytochrome c bound with one tRNA molecule with binding constant of 1.9?×?10(6)?M(-1). The perturbation of cytochrome c structure caused by the binding of tRNA could be affecting its role in mediating apoptosis.     

Interaction of tRNA with Safranal, Crocetin, and Dimethylcrocetin

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants K(safranal) = 6.8 (+/- 0.34) x 10(3) M(-1), K(CRT) = 1.4 (+/- 0.31) x 10(4) M(-1), and K(DMCRT) = 3.4 (+/- 0.30) x 10(4) M(-1). Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.     

The conjugation of chlorambucil with human gamma-globulin.

A method of conjugating chlorambucil with human γ-globulin, with retention of cytotoxic alkylating capacity, using a water soluble carbodiimide as coupling agent, is described.     

Interaction of tRNA with Safranal,Crocetin, and Dimethylcrocetin

Abstract

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants K_safranal = 6.8 (± 0.34) × 10³ M^?1, K_CRT = 1.4 (± 0.31) × 10⁴ M^?1, and K_DMCRT = 3.4 (± 0.30) × 10⁴ M^?1. Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.     

Preparation of synthetic tRNA precursors with tRNA nucleotidyltransferase.

Rabbit liver tRNA nucleotidyltransferase can be used to substitute nucleotides within the -C-C-A sequence of tRNA or to add nucleotides following this sequence. These anomolous reactions of the enzyme have been used to prepare radioactively-labeled synthetic tRNA precursors which mimic the structure of the natural precursors. Under appropriate conditions synthetic precursors of defined structure can be made. In this paper we describe the synthesis of tRNA-C-[14C]U and tRNA-C-C-A-[14C]C-C, which are representative of tRNA precursors containing altered residues within the -C-C-A sequence or with extra residues following the normal 3'terminus. A variety of other possible precursors can also be prepared. These synthetic tRNA precursors have already proved useful for isolation of possible tRNA processing nucleases.     

Interaction of unfolded tRNA with the 3''-terminal region of E. coli 16S ribosomal RNA.

Fragments of tRNA possessing a free TpsiC-loop or a free D-loop form stable complexes with the colicin fragment (1494-1542) of 16S ribosomal RNA from E. coli. The colicin fragment does not bind to tRNA in which the T-loop and the D-loop are involved in tertiary interactions. Colicin cleavage of the 16S rRNA from E. coli is inhibited by aminoacyl-tRNA or tRNA fragments, indicating that a similar interaction may take place on the intact 70S ribosomes. The oligonucleotide d(G-T-T-C-G-A)homologous to the conserved sequence G-T-psi-C-Pu-(m1)A in the TpsiC-region of many elongator tRNAs binds to the conserved sequence U-C-G-mU-A-A-C (1495-1501) of the 16S rRNA. It is suggested that the 3'-end of the 16S rRNA may provide the part of the binding site for the elongator tRNAs on bacterial ribosomes.     

Interaction of eukaryotic threonyl-tRNA synthetase with high-Mr RNAs and tRNA(Thr)

Threonyl-tRNA synthetase of rabbit reticulocytes was purified to homogeneity. We have found that this enzyme can interact not only with cognate tRNA(Thr), but also with high-Mr RNAs. tRNA(Thr) removes rRNA from the complexes with threonyl-tRNA synthetase. On the other hand, rRNA is unable to dissociate tRNA(Thr) from the complexes with the enzyme. Despite its dimeric organization, threonyl-tRNA synthetase is unable to form stable ternary complexes with tRNA(Thr) and rRNA. In the extract of rabbit reticulocytes about one-third of the threonyl-tRNA synthetase molecules are in association with cognate tRNA(Thr) and thus are unable to interact with high-Mr RNAs.     

Interaction of yeast tRNA(Phe) anticodon arm with 40S ribosomal subunit of rabbit liver.

The 15-nucleotide analog of yeast tRNA(Phe) anticodon arm binds cooperatively to two sites of poly(U) programmed 40S ribosome like intact tRNA(Phe). The cooperativity coefficients appeared to be about 4 for tRNA(Phe) and 50 for its anticodon arm. Anticodon arm contributes the majority of free energy of tRNA binding to a programmed 40S ribosomal subunit. The correct codon-anticodon pairing seems to play the key role in the cooperativity origin. Contrary to the anticodon arm template independent binding of the whole tRNA to the small ribosomal subunit is revealed.     

Interaction of tyrosyl aryl dipeptides with S. aureus tyrosyl tRNA synthetase: inhibition and crystal structure of a complex.

Tyrosyl aryl dipeptide inhibitors of S. aureus tyrosyl tRNA synthetase have been identified with IC50 values down to 0.5 microM. A crystal structure of the enzyme complexed to one of the inhibitors shows occupancy of the tyrosyl binding pocket coupled with inhibitor interactions to key catalytic residues.