Mathias Sprinzl

The involvement of 5S RNA in the binding of tRNA to ribosomes

Abstract The tRNA fragment TpψpCpGp was found to bind to 5S RNA. This binding is ten times increased when a specific 5S RNA-protein complex is used. The ability of TpψpCpGp to bind to the complex could be abolished by selective chemical modification of two adenines in 5S RNA. Such 5S RNA, when incorporated into 50S ribosomal subunits, yielded particles with greatly reduced biological activities. From the results presented we conclude that 5S RNA is most likely part of a site with which the TψC-loop of tRNA interacts on the ribosome.

Reaction of the ribose moiety of adenosine and AMP with periodate and carboxylic acid hydrazides

Abstract The dialdehyde (II–V) generated by periodate oxidation of the ribose moiety in adenosine or AMP reacts readily with carboxylic acid hydrazides yielding morpholine derivatives (VI, VII, VIII) which are stable over a wide range of pH and temperature. No side reactions have been observed. This reaction will allow introduction of various substituents into the 3′ end of RNAs, and the resulting modifications would permit investigations on the structure and function of such RNAs.

Aminoacyl-tRNA synthetases from baker's yeast: reacting site of enzymatic aminoacylation is not uniform for all tRNAs.

In previous work we showed that tRNAPhe from baker’s yeast was phenylalanylated exclusively at the 2’hydroxyl group of the terminal ribose by phenylalanyl-tRNA synthetase [ 1,2]. This was based on the observation that after incorporation of 3’deoxyadenosine and 2’deoxyadenosine, respectively, into the terminus of tRNAPhe only tRNAPhe-C-C-3’dA could be aminoacylated whereas tRNAPhe-C-C-2’dA was a competitive inhibitor of the enzyme. The same result was later obtained, by an independent method, for tRNAPhe from Escherichiu coli and from rat liver [3]. These results seemed to be reasonable also from the chemical point of view as suggested by Zamecnik [4] since the 2’hydroxyl group is generally more reactive towards acylation. We then tested tRNAne, tRNASeT*, tRNATvr, and tRNAVa’ from yeast in order to check the general validity of the enzymatic aminoacylation of the more reactive 2’hydroxyl group. We found, however, three classes of tRNAs: those aminoacylated at the 2’hydroxyl group, those aminoacylated at the 3’hydroxyl group and those aminoacylatable at both hydroxyl groups. 2. Materials and methods

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase, and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

Abstract 2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA Phe by tRNA nucleotidyl transferase. tRNA Phe -C-C-2′dA and tRNA Phe -C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA Phe -C-C-3′dA proved to be chargeable.

NMR study on the methyl and methylene proton resonances of tRNAyeastPhe

Summary The proton resonances of 13 methyl groups and 4 methylene groups belonging to 12 modified bases in tRNAyeastPhe were investigated by 220 MHz NMR spectrometry. The chemical shifts and the linewidths at half height of these assigned resonances in tRNA were measured as a function of temperature from 21° to 80°C. The results indicate: (1) The anticodon loop does not associate with other components of the molecule and the side chain of the Y base protrudes out into the solvent; (2) The methyl groups m5C40,49, m2G10, and m1A58 are not near any diamagnetic regions in the native tRNA; (3) The methyl and methylene groups in m22G, T, and hU are magnetically shielded and immobilized to a great extent in the native conformation, implying that these bases are deeply involved in the tertiary structure of tRNA.

Rapid analysis of modified tRNAPhe from yeast by high-performance liquid chromatography: Chromatography of oligonucleotides after RNase T1 digestion on aminopropylsilica and assignment of the fragments based on nucleoside analysis by chromatography on C18-silica

Abstract Aminopropylsilica has been used for the first time as a chromatographic support for the resolution of oligonucleotides produced from RNase T1 digests of native or modified tRNAPhe. The fragments are eluted from this column roughly according to chain length with a combination phosphate methanol gradient in about 1 h. Collection of the resolved oligonucleotides followed by in situ enzymatic digestion and chromatography on C18-silica allows for fast nucleoside analysis and assignment of the oligonucleotide peaks. In addition to the ability to quickly determine the integrity of tRNAs, a comparison of RNAse T1 digests of native tRNAPhe with modified tRNAPhe allows analysis of the position in the tRNA molecule which has undergone modification.

Binding of complementary oligonucleotides to aminoacylated tRNAPhe from yeast

Abstract The effect of the aminoacylation on the structure of tRNA Phe from yeast has been studied by equilibrium dialysis experiments. The association constants of oligomers were determined which were complementary to the dihydrouridine-, the anticodon-, the TψC loop; to the extra-arm and to the 3′-terminus of tRNA Phe -A 73 , tRNA Phe -A-C-C-3′ NH 2 A and Phe-tRNA Phe -A-C-C-3′-NH 2 A in which the phenylalanine is bound by a stable amide bond. The results show that removal of the 3′-terminus or aminoacylation of tRNA Phe from yeast does not cause a gross conformational change of the molecule. However, the aminoacylation renders the 3′-terminus inaccessible to binding complementary oligonucleotides. Based on this finding, it is proposed that the α-amino group of Phe-tRNA Phe -A-C-C-3′ NH 2 A folds back to the 5′-terminal phosphate to form a salt bridge.

Effects of TψCG on the enzymatic binding of eukaryotic and prokaryotic initiator tRNAs to rat liver ribosomes

In 1964 Rosset et al. discovered ribosomal5 S RNA [ 11, which was subsequently sequenced from a number of prokaryotic [2-41 and eukaryotic organisms [S-7]. Comparison of the sequence of KB cell 5 S RNA [5] with that of E. coli [2] lead Forget and Weissman [ 51 to propose that the common 5 S RNA sequence CGAAC would interact with the common GT\kCPu (loop IV) sequence of tRNA [8,9]. More recent data has shown that all 5 S RNAs contain the oligonucleotide or one similar to CGAAC [2-7, lo] . In contrast recent tRNA sequencing experiments have shown that not all tRNAs contain the G’NrCPu oligonucleotide. The exceptions being: 1) eukaryotic initiator tRNA [ 1 l-131 and 2)

Peptidyl transferase center of Escherichia coli ribosomes: interrelations between the substrates.

In 1975 it was shown that cytidine-5’-phosphate stimulates the E. coli ribosomal reaction of pA-Met+-f with Phe-tRNAPhe or C-A-C-C-A-Phe [I]. The stimulating effect of cytidine 5’-phosphate is explained by the tendency of this nucleotide to be bound to the area of the donor site of the peptidyl transferase centre occupied in an ordinary process by the penultimate 3’-terminal nucleotide of peptidyl-tRNA [2-41. By an identical mechanism cytidine 5’-phosphate also stimulates the reactions catalyzed by 50 S subunits of E. coli ribosomes [5] and by rat liver ribosomes [6]. All these facts indicate some allosteric effects taking place in the peptidyl transferase centre as a result of the occupancy of the ribosomal Aand P-site. Several experiments have been reported which indicate that the activity of one ribosomal site can be influenced by the reaction taking place in the other site of the ribosomes (reviewed [7]). Among them the dependence of activity of puromycin [8] and other model acceptors [9] upon the nature of peptide donor should be mentioned. The peptidyl-tRNA bound to the donor site stimulates binding of the complex aminoacyl-tRNA + EF-I t GTP to rabbit

Crystallisation of yeast phenylalanine transfer RNA: Polymorphism and studies of sulphur-substituted mercury binding derivatives

Abstract Crystallization procedures and X-ray crystallographic investigations are reported for eight different crystal forms of native yeast tRNAPhe. The crystals belong to the space groups monoclinic P21 and C2, orthorhombic P21 221 and C2221, hexagonal P62 22 with two different unit cells, rhombohedral R32, and cubic I41 32. The experimental conditions under which the various forms can be obtained are defined and the question is discussed as to whether these crystal forms reflect different conformations of the tRNA molecule itself. Further, yeast tRNAPhe was modified chemically with the objective of obtaining heavy atom substitution suitable for X-ray crystal structure analysis: s4 UMP can be incorporated into position 75 of tRNAPhe by tRNA nucleotidyl transferase yielding tRNAPhepCps4 U. This modified tRNA, tRNAPhepCpCspA, and tRNAPhepCps2 CpA crystallised in suitable specimens isomorphous with native yeast tRNAPhe while the iodine analogue tRNAPhe p(i5Cpi5 CpA crystallised in small, as yet uncharacterized crystals. Attempts have been made to obtain mercury derivatives by treating the sulphur-containing tRNAs with mercurials.