Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

RNA-ligant interactions. (I) Magnesium binding sites in yeast tRNAPhe.

X-ray crystallagraphic studies studies indicate that there are at least four site-specifically bound hydrated Mg2+ ions, [Mg(H2O)n]2+, in yeast tRNAPhe. The size and the octahedral coordination geometry, rather than the charge, of [Mg(H2O)N]2+ appear to be the primary reasons for the specificity of magnesium ions in site-binding and in the stabilization of the tertiary structure of tRNA.     

Lead-catalyzed cleavage of yeast tRNAPhe mutants

Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure. Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48. The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.     

Specific heavy metal labeling of the 3-'terminus of phosphorothioate modified yeast tRNAPhe.

Yeast tRNAPhe containing a phosphorothioate modified -CS-CS-A terminus binds two moles of chloroterpyridineplatinum(II). This result was determined by titrating the tRNA with [3H](terpy)PtCl] Cl, removing excess platinum by cation exchange chromatography, and determining the amount of bound platinum by radiocounting techniques. It has thus been established that adjacent phosphorothioate modified nucleotides can be labeled with an electron dense stain, a necessary requirement for electronmicroscopic sequencing of polynucleotides to become practical.     

Fluorescent derivatives of yeast tRNAPhe.

The preparation of four fluorescent derivatives of tRNAPhe (yeast) and their characterization by chemical, spectroscopic, and biochemical methods is described. The derivatives are prepared by replacing wybutine (position 37 in the anticodon loop) or NaBH4-reduced dihydrouracil (positions 16/17 in the hU loop) with ethidium or proflavine; they are isolated by reversed-phase chromatography (RPC-5). All tRNAPhe-dye derivatives are aminoacylated by yeast phenylalanyl-tRNA synthetase to at least 80% of the charging capacity of the unmodified tRNAPhe with an unchanged Km (0.2 mucroM) and a V lowered by 30--50%. They exhibit good to excellent activity in the aminoacylation assay from synthetase from Escherichia coli. It is concluded that the insertion of the dyes does not seriously disturb essential elements of the native tRNAPhe structure. The dyes are bound via N-ribosylic linkages. The appearance of isomeric tRNAPhe-ethidium derivatives is attributed to the involvement of the different amino groups of ethidium in the condensation. In addition, there are indications for the existence of alpha and beta anomers of the tRNA-dye compounds. The dyes are rigidly fixed to their position in the tRNA molecule by stacking interactions with the neighboring bases. The ethidium probes show Mg2+-induced changes of the tRNA conformation which are paralleled by changes of the rate of aminoacylation. On the basis of this observation it is hypothesized that conformational flexibility of the tRNA molecule is a functionally important feature of the tRNA structure.     

Chemical conversion of cytidine residues into 4-thiouridines in yeast tRNAPhe. Determination of the modified cytidines

Treatment of yeast phenylalanine tRNA with pressurized hydrogen sulfide results in conversion of cytidine residues into 4-thiouridine residues. Under conditions leading to an average modification of one cytidine per tRNA molecule 9 positions are thiolated. The 4-thiouridine residues are distributed along the tRNA molecule. Four of the reactive cytidines are located in single-stranded regions: Cm32 , C60 , C74 and C75 . The five others are located in base pairs: C2, C27, C56 , C61 and C63 . Importance of replacement of an amino group by a thiol group on hydrogen bonding and on biological activity of the modified tRNA is discussed.     

Fluorescence studies of the binding of a yeast tRNAPhe derivative to Escherichia coli ribosomes.

The equilibrium binding of a highly fluorescent derivative of yeast tRNA^Phe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 10⁹m^?1 and (3.4 ± 0.2) × 10⁷m^?1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.     

Further refinement of the structure of yeast tRNAPhe.

We have refined the monoclinic crystal structure of yeast tRNA^Phe against a complete set of X-ray data at 2.5 Å resolution, using real-space refinement and a combination of energy minimisation and crystallographic least-squares. This refinement has allowed us to define the conformation of residue D16, and to make corrections to Y37 and A76. We have found an additional magnesium binding site (making a total of four), a number of water molecules, and a possible spermine molecule.A revised list of torsion angles is given.     

Selective binding of amino acid residues to tRNAPhe

The interaction of amino acid amides with tRNAPhe was studied by measurements of the Wye base fluorescence. Binding of phenylalanine-, tyrosine- and tryptophan-amides leads to considerable quenching, whereas the amides of e.g. glycine and leucine do not induce quenching under the same conditions. Binding constants at 0.13 M salt - 100 M-1 for Phe-, 110 M-1 for Tyr- and 300 M-1 for Trp-amide - are about a factor of 6 higher than those evaluated from independent measurements for binding to simple single-stranded polynucleotides; the corresponding factor is 10 for double-stranded polynucleotides. Since the apparent enthalpy changes derived from measurements at different temperatures remains relatively low (-9 to -20 kJ/mol), the increased affinity appears to be mainly due to an increase of the entropy changes. Titration experiments performed in the presence of Mg2+ indicate cooperative interactions of the aromatic residues with the anticodon loop that are consistent with preferential binding to one of two loop conformations. Measurements of binding constants at different pH-values indicate the protonation of a tRNA residue in the tryptophanamide-tRNAPhe complex characterised by a pK value of about 7.0.     

A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.     

Evidence for the existence of an expressed minor variant tRNAPhe in yeast

Two expressed brewer's yeast tRNAsPhe, a major and a minor one, have been purified and sequenced. The major tRNAPhe corresponds to the already known tRNAPhe, whereas the minor one differs from the former in the substitution of T6-A67 by C6-G67 base pair in the "acceptor stem". The minor tRNAPhe contaminates all preparations of yeast tRNAPhe except those prepared by polyacrylamide gel electrophoresis.     

Quantitative aspects of metal ion binding to certain transfer RNA anticodon loop modified nucleosides

Magnesium and manganese ions bind strongly to the unusual transfer RNA anticodon loop nucleotides, N-[(9-beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]-L-threonine 5'-monophosphate (pt6A) and uridine-5-oxyacetic acid 5'-monophosphate (pV). Potentiometric measurements have shown that the delta G for metal ion-pt6A complex formation is 2-3-times more exothermic than for AMP. Electron-nuclear longitudinal dipolar relaxation data yielded manganese-ligand atom distances which permit a three-dimensional construct of the complex in which metal is coordinated to the phosphate, carboxylate of the threonine side-chain (with the nucleotide in the anti glycosidic conformation) and N7 of the adenine ring. Similarly, manganese binds strongly to pV, involving phosphate and carboxylate functions. It is possible that a facet of the functional role of these unusual residues is to chelate magnesium ions and in so doing permit optimum anticodon loop conformational stability and stability of tRNA-mRNA-ribosome complexes.     

The influence of spermine on the structural dynamics of yeast tRNAPhe

A tRNAPhe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588-4599) in the tRNA derivative the ethidium is present in three states (T1-T3) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10-100 ms) between T1 and T2, and a slow one (100-1000 ms) between T2 and T3. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T3, as does Mg2+, but the final ratio [T2]/[T1] obtained with spermine is higher than with Mg2+, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

Local folding coupled to RNA binding in the yeast ribosomal protein L30.

The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regulates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA. The three-dimensional structures of L30 in the free (f L30) and the pre-mRNA bound (b L30) forms have been solved by nuclear magnetic resonance spectroscopy. Both protein structures contain four alternating alpha-helices and four beta-strands segments and adopt an overall topology that is an alphabetaalpha three-layer sandwich, representing a unique fold. Three loops on one end of the alphabetaalpha sandwich have been mapped as the RNA binding site on the basis of structural comparison, chemical shift perturbation and the inter-molecular nuclear Overhauser effects to the RNA. The structural and dynamic comparison of f L30 and b L30 reveals that local dynamics may play an important role in the RNA binding. The fourth helix in b L30 is longer than in f L30, and is stabilized by RNA binding. The exposed hydrophobic surface that is buried upon RNA binding may provide the energy necessary to drive secondary structure formation, and may account for the increased stability of b L30.     

NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].     

Circular dichroism study of modified nucleosides

CD study of four modified nucleosides, constituents of tRNA molecules, revealed that 2-thio-5-methyluridine and 5-methyluridine in aqueous solution, 0.1N HCl, and organic solvents essentially occur in an anti-conformation. 5-Methylcytidine also occurs in an anti-conformation similar to cytidine in aqueous solution and organic solvents, while 2-thiocytidine dihydrate appears to occur in an anti-conformation. It is stressed that the CD data of thionucleosides might be applied to the successfully conformational analysis of tRNA molecules.