Neurospora crassa mitochondrial transfer RNAs.

Total mitochondrial tRNA from Neurospora crassa was characterized by base composition analysis, one- and two-dimensional gel electrophoreses and reversed-phase chromatography on RPC5. The guanosine + cytidine content was about 43%, as compared to 60% for cytoplasmic tRNA. The modified nucleoside content was low and about the same as that of total yeast mitochondrial tRNA, though the G + C content is very different. We found psi, T, hU, t6A, m1G, M2G, m22G. Neither the eukaryotic "Y" base, nor the prokaryotic s4U were present. On two-dimensional polyacrylamide gel electropherograms about 25 species were separated. One species for phenylalanine, two for leucine and two for methionine could be located. Neurospora crassa mitochondrial tRNA does not hybridize with yeast mitochondrial DNA.     

Effects of lymphocyte activation on transfer RNAs

The influences of mitogen activation on the functional capacity of rat splenic tRNAs were evaluated. The specific amino acid acceptor activity, pmol of a specific amino acid accepted per nmol of tRNA, of isolated splenic tRNAs from in vivo Concanavalin A (37 h)-treated rats were up to 8 times the specific amino acid acceptor activities of splenic tRNAs from control rats. Control splenic tRNAs were treated with purified liver tRNA nucleotidyltransferase in vitro to repair the 3'[CCA] terminus of tRNAs, and subsequently assayed in an aminoacylation reaction. The specific amino acid acceptor activities were slightly increased over those tRNAs not repaired with tRNA nucleotidyltransferase, indicating the presence of a low level of defective but repairable tRNAs in the control rat spleen. Furthermore, our results indicate that cyclosporin A (inhibitor of lymphocyte activation) blocks the Concanavalin A stimulation of tRNA charging ranging from 16 to 93%.     

Regulatory RNAs derived from transfer RNA?

Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.     

Affinity chromatography of tyrosine transfer RNAs.

Ricinus communis agglutinin, a lectin from castor beans has an affinity for β-d-galactose and tyrosine tRNAs of mammalian tissues have galactose in gal-Q base of their anticodons. We have studied interaction between tyrosine tRNAs and this lectin immobilized on solid supports using spacer arms of different lengths. Tyrosine tRNAs are separated from nineteen other tRNAs of bovine liver by affinity chromatography using the lectin immobilized to an agarose matrix. The results indicate that a spacer arm length of 10 Å between the agarose bead and the lectin gives the best separation. Two tyrosine tRNA isoacceptors are separated from each other and from other tRNAs in one step using this affinity column chromatography.     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

Functional interactions by transfer RNAs in the ribosome

Recent X-ray crystal structures of the ribosome have revolutionized the field by providing a much-needed structural framework to understand ribosome function. Indeed, the crystal structures rationalize much of the genetic and biochemical data that have been meticulously gathered over 50 years. Here, we focus on the interactions between tRNAs and the ribosome and describe some of the insights that the structures provide about the mechanism of translation. Both high-resolution structures and functional studies are essential for fully appreciating the complex process of protein synthesis.     

Chloroplast transfer RNAs and tRNA genes of wheat

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids. Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region. A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.     

Evolution of methionine initiator and phenylalanine transfer RNAs

Summary Sequence data from methionine initiator and phenylalanine transfer RNAs were used to construct phylogenetic trees by the maximum parsimony method. Although eukaryotes, prokaryotes and chloroplasts appear related to a common ancestor, no firm conclusion can be drawn at this time about mitochondrial-coded transfer RNAs. tRNA evolution is not appropriately described by random hit models, since the various regions of the molecule differ sharply in their mutational fixation rates. Hot mutational spots are identified in the TC, the amino acceptor and the upper anticodon stems; the D arm and the loop areas on the other hand are highly conserved. Crucial tertiary interactions are thus essentially preserved while most of the double helical domain undergoes base pair interchange. Transitions are about half as costly as transversions, suggesting that base pair interchanges proceed mostly through G-U and A -C intermediates. There is a preponderance of replacements starting from G and C but this bias appears to follow the high G + C content of the easily mutated base paired regions.     

Electrical potential of transfer RNAs: codon-anticodon recognition

Calculations of the electrostatic potentials were made around yeast elongator phenylalanine, aspartate tRNAs, and yeast initiator methionine tRNA in aqueous solution at physiological ionic strength. The calculations were carried out with a finite difference algorithm for solving the nonlinear Poisson-Boltzmann equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape, and the different polarizabilities of the solvent and the tRNA. The initiator tRNA is surrounded by uniformly spaced contours of negative potential. The elongator tRNAs are also surrounded by a similar contour pattern except in the anticodon region where there is a pronounced "hole" in the potential surface. This hole is caused by an invagination of the potential contours, which also results in an increase in the local field strength. The effect of this hole is that the anticodon region in the elongator tRNAs is the least negative, or conversely the most positive, region of the molecule. This hole, which is not found when simple Coulombic potentials are used, is due both to the structure of the elongator tRNA anticodon loops and to the different polarizabilities of the solvent and tRNA. The existence of the potential hole in elongator tRNAs may account in part for their ability to associate with other negatively charged macromolecules, in particular mRNA. Moreover, it suggests that the anticodon loop of elongator tRNAs is the energetically most favorable point of approach of mRNA to tRNA.     

Recent results on how aminoacyl transfer RNA synthetases recognize specific transfer RNAs

Summary Aminoacyl tRNA synthetases discriminate between tRNA species by a highly specific mechanism. Physical and chemical studies indicate that the synthetases bind along and around the inside of the three-dimensional L-shaped tRNA structure. Studies of mutant tRNAs that affect synthetase interaction tend to confirm this conclusion. However, in contrast to proteins that recognize a specific block of contiguous nucleotide units (e.g., repressors, restriction enzymes, etc.), synthetases appear to interact with spatially disperse elements of the structure. Available evidence suggests that tRNA binding clefts on various synthetases may be roughly similar, with specificity being achieved by the choice of amino acid residues in a few critical positions in the tRNA binding clefts. With this idea in mind, it should be possible to introduce amino acid substitutions into the binding clefts and thereby change tRNA recognition specificity. This has been attempted (by genetic manipulations) and a mutant alanine tRNA synthetase with altered tRNA recognition has been isolated. This enzyme can attach alanine to isoleucine specific tRNA. When presented with valine specific tRNA, a tRNA similar in some structural features to the isoleucine specific tRNA, or with the structurally quite different tyrosine specific tRNA, no significant aminoacylation occurs. Thus, a precise specificity alteration can occur through mutation; this result supports the idea of similarities in synthetase binding clefts, with specificity being achieved by the positioning of amino acids at critical positions in these clefts. Finally, further data have been obtained on the issue of possible transient covalent bond formation between synthetases and tRNAs, as a critical part of the interaction.Abbreviations tRNA^x a tRNA specific for the amino acid - x where x is given the standard 3 letter abbreviation