Modification of tRNA 1 Val from yeast with monoperphthalic acid]

A method is proposed for analysis of natural and chemically modified polynucleotides which consists in enzymatic conversion of the polymer or oligomer into nucleosides followed by cation-exchange chromotography on the microcolumns. By using the method developed it was shown that after treatment of the yeast tRNAVal and tRNAPhe with monoperphthalic acid N-oxides of adenosine and cytidine were formed. Poly (U, G) was not modified at a measurable extent whereas GMP was decomposed. In tRNAVal (yeast)the adenosines and cytosines of the anticodon loop and 3'-end are most reactive; it is the case for the C17 of the diHU-loop as well. These data are in agreement with the results obtained for tRNA modification with other reagents and for limited enzymatic hydrolysis of the tRNAVal. The limitations of the reaction of the monoperphthalate with nucleic acids are briefly discussed.     

The primary structure of tRNA Val 2a from baker's yeast]

The primary structure of tRNAVal2a from baker's yeast has been determined. The general methods of the investigation are presented. Twenty six distinguished points can be noted in the tRNAVal2a and tRNA1Val from baker's yeast. The anticodon region of tRNAVal2a is represented by the sequence NAC, where N corresponds to a uridine analogue nucleoside of unknown structure. The comparison of primary structures of tRNAVal2a, tRNAVal2a, tRNA1Val from E. coli and tRNAVal2a and tRNA1Val from baker's yeast is analysed.     

Synthesis of fragments of the D-branch of yeast valine tRNA1 and their analogs

Nonanucleotide GpUpCpUpApGpDpCpGp corresponding to the fragment 10-18 of the yeast tRNA1Val with pseudouridine-13 replaced by uridine has been synthesized. Dihydrouridine derivatives were studied as substrated of ribonucleases with various specificity and of RNA ligase.     

Structure of the yeast tRNA m7G methylation complex

Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.     

Interactions of some naturally occurring cations with phenylalanine and initiator tRNA from yeast as reflected by their thermal stability

The thermal unfolding of phenylalanine and initiator tRNA from yeast was investigated over a broad range of solution conditions by differential ultraviolet absorption at 260 nm. Under most conditions, the initiator tRNA exhibits two clearly separated transitions in its differential melting curve which were assigned to unfolding of tertiary and secondary structure elements, respectively. The tertiary transition of this tRNA and the overall transition observed for tRNAPhe do not show a maximum in a curve of Tm values plotted as a function of [Na+]. Such a maximum is usually observed for other nucleic acids at about 1 M Na+. In the presence of 5 mM of the divalent cation Mg2+ (or Ca2+), an overall destabilization of the tRNAs is observed when increasing the sodium concentration. The largest fall in Tm (approximately 15 degrees C) is observed for the tertiary transition of the initiator tRNA. Among various cations tested the following efficiency in the overall stabilization of tRNAPhe is observed: spermine greater than spermidine greater than putrescine greater than Na+ (approximately NH4+). Mg2+ is most efficient at concentrations above 5 mM, but below this concentration spermine and spermidine appear to be more efficient. The same hierarchy in stabilizing power of the polyamines and Na+ is observed for both transitions of the initiator tRNA. However, when compared with Mg2+, the polyamines are far less capable of stabilizing the tertiary structure. In contrast, spermine and spermidine are slightly better than Mg2+ in stabilizing the secondary structure. At increasing concentrations of the polyvalent cations (at fixed [Na+] ) the Tm values of the tRNAs attain a constant value.     

High-performance liquid chromatographic analysis of crystals of tRNA, aminoacyl-tRNA synthetase, and their complex

High-performance liquid chromatography on an ion exchanger column was successfully used for a rapid biochemical analysis of crystals of yeast tRNAAsp and aspartyl-tRNA synthetase as well as cocrystals formed by the synthetase and the tRNA.     

High-resolution nmr study of yeast tRNA and the native and denatured conformers of yeast tRNA

The high-resolution nmr spectrum of baker's yeast tRNA, a recently sequenced non-denaturable tRNA, has been compared with the spectra of the native and denatured conformers of the closely related species tRNA. Because of the presence of many common base pairs in the different tRNA's, it is possible to assign most of the low-field resonances to specific secondary-structure base pairs. A comparison of the observed positions of the various resonances with those predicted by a semiempirical ring-current shift theory shows a root-mean-square deviation of 0.14, 0.11, and 0.12 ppm for tRNA (native), and tRNA (denatured), respectively. These results support the ring-current shift theory currently used to interpret the low-field nmr spectra of the tRNA molecules. Differences between the predicted and observed positions of some resonances provide new evidence for higher order effects such as shifts from second nearest neighbors, anomalous shifts exerted by G·U base pairs, and tertiary-structure effects. A model that was previously proposed for the denatured conformer of tRNA is also supported.