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.     

Compilation of tRNA sequences and sequences of tRNA genes.

Sequences of 3279 sequences of tRNA genes and tRNAs published up to December 1996 are included in the compilation. Alignment of the sequences, which is most compatible with the tRNA phylogeny and known three-dimensional structures of tRNA, is used. Sequences and references are available under http://www.uni-bayreuth. de/departments/biochemie/trna/     

Compilation of tRNA sequences.

This compilation presents in a small space the tRNA sequences so far published in order to enable rapid orientation and comparison. The numbering of tRNAPhe from yeast is used as has been done earlier (1) but following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (2) (Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe, the only structure known from X-ray analysis. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguously analyzed. Rare nucleosides are named according to the IUPAC-IUB rules (for some more complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3--7). Mutant tRNAs are dealt with in a separate compilation prepared by J. Celis (see below). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation.     

Compilation of tRNA sequences.

This compilation presents in a small space the tRNA sequences so far published. The numbering of tRNAPhe from yeast is used following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (1,2;Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguosly analyzed. Rare nucleosides are named according to the IUPACIUB rules (for complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3-7). Mutant tRNAs are dealt with in a compilation by J. Celis (8). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation.     

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.     

The identification of the tRNA substrates for the supK tRNA methylase.

Purified preparations of the tRNA methylase deficient in supK strains of Salmonella typhimurium transfer methyl groups from S-adenosylmethionine (SAM) to at least two tRNA species, an alanine tRNA and a serine tRNA. The identity of the tRNA substrates for this enzyme was determined by a change in the elution position of the methyl-labeled tRNA from BND-cellulose columns before and after aminoacylation with a specific amino acid followed by derivatization of the free primary amino group with phenoxy- or naphthoxyacetate. The radioactive methyl group enzymatically added to these tRNAs is both acid and base labile and can be hydrolyzed to a volatile product at pHs above 7.5 and also at pH 1. The methylated 3'-nucleotide isolated from digested tRNA is a pyrimidine derivative and chromatographs like a modified uridylic acid. Its identity has not been established, but it is likely that it corresponds to the methyl ester of V, uridin-5-oxyacetic acid.     

The evolution of multi-isoacceptor tRNA families. Sequence of tRNA Leu CAA and tRNA Leu CAG from Anacystis nidulans

Two leucine tRNAs from the cyanophyte Anacystis nidulans have been isolated, and their complete nucleotide sequences have been determined by combining data from oligonucleotide fingerprints and sequencing gels. The two sequences are 87 nucleotides long, have the anticodons CAA and CAG, and differ from each other at a total of 28 positions. They have been compared to other known tRNA Leu sequences and incorporated into a phylogenetic tree comprising prokaryotic and chloroplastic tRNA Leu sequences. Mutations inferred from the tree show that some parts of the tRNA molecule are highly variable (the extra arm and the acceptor stem) while others are much more conserved (the D and T arms). The topology of the tree supports the idea that blue-green algae and chloroplasts share a common prokaryotic ancestor and show a basic divergence between XAA and XAG anticodon-containing tRNAs, suggesting that these two subfamilies result from an ancient gene duplication. Finally, comparison of this phylogenetic tree with those of other multi-isoacceptor tRNA families shows no common scheme, which may be due to independent refinement of codon-reading patterns in different tRNA families.     

tRNA prefers to kiss.

Six RNA aptamers that bind to yeast phenylalanine tRNA were identified by in vitro selection from a random-sequence pool. The two most abundantly represented aptamers interact with the tRNA anticodon loop, each through a sequence block with perfect Watson-Crick complementarity to the loop. It was possible to truncate one of these aptamers to a simple hairpin loop that forms a classical 'kissing complex' with the anticodon loop. Three other aptamers have nearly complete complementarity to the anticodon loop. The sixth aptamer has two sequence blocks, one complementary to the tRNA T loop and the other to the D loop; this aptamer binds better to a mutant tRNA that disrupts the normal D-loop/T-loop tertiary interaction than to the wild-type tRNA. Selection of complements to tRNA loops occurred despite an attempt to direct binding to tertiary structural features of tRNA. This serves as a reminder of how special the RNA-RNA interactions are that are not based on complementarity. Nonetheless, these aptamers must present the tRNA complement in some special structural context; the simple single-strand complement of the anticodon loop did not bind tRNA effectively.     

Interaction of chlorambucil with tRNA.

Preincubation of purified mixed tRNAs from Escherichia coli K12-MO with 2.94 mM chlorambucil (CAB) for 2 h at 37 degrees C results in the inhibition of the capacity of mixed tRNAs to accept alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine by 100, 71, 100, 100, 100, 95, 32, 88, 36, 26, 96, 78, 44, 31, 34, 98, 38, and 17% respectively. Preincubation of tRNA with 0.75 mM and 0.29 mM CAB inhibited aminoacylation by aspartic acid to the extent of 69 and 17% respectively. CAB has no apparent effect upon the capacity of ATP to function in the formation of aminoacylated tRNALeu.     

Aminoacyl-tRNA synthetase-induced cleavage of tRNA.

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.