Investigation of immunoadsorbent efficiency and capacity for the isolation of tRNAs containing N6-(delta 2-isopentenyl)adenosine derivatives

Antibodies directed to modified nucleosides recognize the nucleoside (antigen) when it is present in an intact tRNA molecule. The general application of anti-nucleoside immunoadsorbent chromatography, however, has been greatly impeded by the apparent inefficiency and low capacity of conventional immunoadsorbents for transfer RNA. Antibodies specific for isopentenyladenosine (i6A) were employed to investigate the efficacy of various immunoadsorbents with respect to immobilization of antibody protein and with respect to their ability to bind i6A-containing tRNAs. Biologically active anti-i6A was recovered in high yield (80-88%) by affinity chromatography on i6A-adipate-Sepharose 4B or i6A-butane diglycidyl ether-Sepharose 4B using either 15% pyridine in phosphate-buffered saline or 0.2 M acetic acid as eluents. The binding capacity of various anti-i6A antibody immunoadsorbents was evaluated. While both anti-i6A antibody-protein A-agarose-iminothiolane (ITL) and anti-i6A antibody-protein A-agarose-dimethyl suberimidate showed a high capacity for i6A-tRNA, the latter column is much less efficient with respect to antibody immobilization. Under optimal conditions, the ITL immunoadsorbent binds 5-6 nmol of i6A/mg of antibody protein. With respect to bulk tRNA, 1 mg of antibody protein (ITL immunoadsorbent) binds all of the i6A-tRNA in a 1-mg sample.     

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs. II. Analysis of 7-methylguanosine in mitochondrial and cytosolic aminoacyl-transfer RNAs.

The 7-methylguanosine (m7G) content of two individual mitochondrial tRNAs, labelled in the aminoacyl moiety was assayed by the specific cleavage of the tRNA at this nucleotide followed by electrophoretic analysis to identify the 3'-terminal fragment of the tRNA. Neither Syriam hamster mitochondrial tRNALeu nor tRNAMet were found to contain m7G. In contrast, cytosolic tRNAMetS were cleaved indicating the presence of m7G, apparently 27--28 and 29 nucleotides from their 3' terminus. Cystolic tRNALeu was not cleaved. These results are discussed in relationship to the reported low content of methylated nucleosides in mitochondrial 4 S RNA.     

Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons

Mutant tRNAs containing an extra nucleotide in the anticodon loop are known to suppress +1 frameshift mutations, but in no case has the molecular mechanism been clarified. It has been proposed that the expanded anticodon pairs with a complementary mRNA sequence (the frameshift sequence) in the A site, and this quadruplet "codon-anticodon" helix is translocated to the P site to restore the correct reading frame. Here, we analyze the ability of tRNA analogs containing expanded anticodons to recognize and position mRNA in ribosomal complexes in vitro. In all cases tested, 8 nt anticodon loops position the 3' three-quarters of the frameshift sequence in the P site, indicating that the 5' bases of the expanded anticodon (nucleotides 33.5, 34, and 35) pair with mRNA in the P site. We also provide evidence that four base-pairs can form between the P-site tRNA and mRNA, and the fourth base-pair involves nucleotide 36 of the tRNA and lies toward (or in) the 30 S E site. In the A site, tRNA analogs with the expanded anticodon ACCG are able to recognize either CGG or GGU. These data imply a flexibility of the expanded anticodon in the A site. Recognition of the 5' three-quarters of the frameshift sequence in the A site and subsequent translocation of the expanded anticodon to the P site results in movement of mRNA by four nucleotides, explaining how these tRNAs can change the mRNA register in the ribosome to restore the correct reading frame.     

Recombinant photoreactive tRNA molecules as probes for cross-linking studies

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.     

Antibody nucleic acid complexes. Immunospecific retention of N6-methyladenosine-containing transfer ribonucleic acid.

Antibodies specific for N6-methyladenosine (m6A) were immobilized on Sepharose and the resulting immunoadsorbent was tested for its ability to retain those Escherichia coli tRNAs containing the antigenic hapten, i.e., m6A. Results obtained with [32P]PO4- and [methyl-3H]-methionine-labeled tRNAs indicated that approximately 3 to 5% of the radioactive RNA was retained by the immunoadsorbent. Under identical conditions, but in the presence of m6A (1 mg/mL), less than 0.2% of the radioactivity was retained. Subsequent characterization of the retained tRNA via (a) analysis of methyl-3H-labeled, methylated nucleosides, (b) two-dimensional gel electrophoresis, and (c) analysis of the retention of [3H]aminoacyl-tRNA species led to the conclusion that the anti-m6A/Sepharose adsorbent quantitatively and exclusively retained a single tRNA species containing m6A, namely, tRNAVal.     

The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification.

Selenocysteine is cotranslationally incorporated into selenoproteins in a unique pathway involving tRNA mediated suppression of a UGA nonsense codon (1-3). The DNA sequence of the gene for this suppressor tRNA from Escherichia coli predicts unusual features of the gene product (4). We determined the sequence of this serine tRNA (tRNA(UCASer]. It is the longest tRNA (95 nt) known to date with an acceptor stem of 8 base pairs and lacks some of the 'invariant' nucleotides found in other tRNAs. It is the first E. coli tRNA that contains the hypermodified nucleotide i6A, adjacent to the UGA-recognizing anticodon UCA. The implications of the unusual structure and modification of this tRNA on recognition by seryl-tRNA synthetase, by tRNA modifying enzymes, and on codon recognition are discussed.     

tRNAs are imported into mitochondria of Trypanosoma brucei independently of their genomic context and genetic origin.

The mitochondrial genome of Trypanosoma brucei does not encode any identifiable tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. In order to analyse the signals which target the tRNAs into the mitochondria, an in vivo import system has been developed: tRNA variants were expressed episomally and their import into mitochondria assessed by purification and nuclease treatment of the mitochondrial fraction. Three tRNA genes were tested in this system: (i) a mutated version of the trypanosomal tRNA(Tyr); (ii) a cytosolic tRNA(His) of yeast; and (iii) a human cytosolic tRNA(Lys). The tRNAs were expressed in their own genomic context, or containing various lengths of the 5'-flanking sequence of the trypanosomal tRNA(Tyr) gene. In all cases efficient import of each of the tRNAs was observed. We independently confirmed the mitochondrial import of the yeast tRNA(His), since in organello [alpha-32P]ATP-labelling of the 3'-end of the tRNA was inhibited by carboxyatractyloside, a highly specific inhibitor of the mitochondrial adenine nucleotide translocator. Import of heterologous tRNAs in their own genomic contexts supports the conclusion that no specific targeting signals are necessary to import tRNAs into mitochondria of T. brucei, but rather that the tRNA structure itself is sufficient to specify import.     

The T-stem determines the cytosolic or mitochondrial localization of trypanosomal tRNAsMet

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNA(Met) is closely related to the imported elongator tRNA(Met). Thus, the distinct localization of the two tRNAs(Met) must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAs(Met). Furthermore, it was shown that the tRNA(Met) T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNA(Met) indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAs(Met) into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNA(Met).     

Lysine tRNAs from rat liver: lysine tRNA sequences are highly conserved

The two major lysine tRNAs from rat liver, tRNA2Lys and tRNA5Lys, were sequenced by rapid gel or chromatogram readout methods. The major tRNA2Lys differs from a minor form only by a base pair in positions 29 and 41; both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Although highly related, the major tRNA2Lys and tRNA5Lys differ in four base pairs and four unpaired nucleotides, including the first position of the anticodons, but have the same base pair in positions 29 and 41. The three tRNAs maintain a m2G-U pair in the acceptor stem. Detection of this m2G is in contrast to other reports of lysine tRNAs. Sequences of lysine tRNAs are strongly conserved in higher eukaryotes.     

supG and supL in Escherichia coli code for mutant lysine tRNAs+.

We have determined the nucleotide sequences of lysine tRNAs isolated from strains containing one or the other of two Escherichia coli ochre suppressors, supG and supL. Each strain, besides producing wild-type lysine tRNA, has a mutant lysine tRNA species that apparently can read the polypeptide chain termination codons UAA and UAG. The mutant tRNAs from supG and supL strains are identical. In each case the suppressor tRNA has an A36 for U36 nucleotide substitution. Furthermore, the hypermodified nucleoside at position 37 has been changed from t6A to ms2i6A.     

Nucleotide sequence of non-initiator methionine tRNA from Bacillus subtilis

Non-initiator methionine tRNA (tRNAMet) was purified from Bacillus subtilis W 168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be p-G-G-C-G-G-U-G-U-A-G-C-U-C-A-G-C-G-G-C-D-A-G-A-G-C-G-U-A-C-G-G-U-U-C-A-U-m6A-C-C-C -G-U-G-A-G-G(m7G)-U(D)-C-G-G-G-G-G-T-psi-C-G-A-U-C-C-C-C-U-C-C-G-C-C-G-C-U-A-C- C-A-OH. The nucleosides of G46 and U47 were partially modified to m7G and D, respectively. The nucleotide sequence shows a unique feature that the position adjacent to 3'-end of the anticodon C-A-U is occupied by m6A, not by t6A, although the tRNAMet belongs to a groups of tRNAs which recognize codons starting with A.     

Nucleotide correlations that suggest tertiary interactions in the TV-replacement loop-containing mitochondrial tRNAs of the nematodes, Caenorhabditis elegans and Ascaris suum.

In the predicted secondary structures of 20 of the 22 tRNAs encoded in mitochondrial DNA (mtDNA) molecules of the nematodes, Caenorhabditis elegans and Ascaris suum, the T psi C arm and variable loop are replaced with a loop of 6 to 12 nucleotides: the TV-replacement loop. From considerations of patterns of nucleotide correlations in the central regions of these tRNAs, it seems highly likely that tertiary interactions occur within five sets of binary and ternary combinations of nucleotides that correspond in location to nucleotides known to be involved in tertiary interactions in yeast tRNA(Phe) and other standard tRNAs. These observations are consistent with the nematode TV-replacement loop-containing mt-tRNAs being folded into a similar L-shaped functional form to that demonstrated for standard tRNAs, and for the bovine DHU (dihydrouridine) arm replacement-loop-containing mt-tRNA(Ser(AGY)). However, the apparent occurrence in nematode mt-tRNAs of tertiary bonds common to standard tRNAs contrasts with the situation in bovine mt-tRNA(Ser(AGY)) where the functional form is dependent on an almost unique set of tertiary interactions. Because three of the proposed conserved tertiary interactions in the nematode mt-tRNAs involve nucleotides that occur in the variable loop in standard tRNAs, it seems more likely that in nematode mt-tRNAs it is the T psi C arm rather than the variable loop that has undergone the greatest proportional decrease in nucleotide number.     

A unique method utilizing antinucleotide antibodies for evaluating changes in the levels of modified nucleosides of tRNAs from crude extracts of whole cells.

The utilization of antibodies directed toward modified nucleosides in evaluating changes in the levels of certain modified nucleosides in transfer RNA is reported. Antibodies directed toward the N6-(delta 2-isopentenyl)adenosine modification were used in this model system with a mutant strain of Escherichia coli designated ipaA. The procedure is rapid, sensitive, and specific. In addition, it does not depend on the existence of an in vitro remodification system or any radiochemical labeling of the tRNA. By varying the extraction technique, the method could be applied to procaryotic or eukaryotic cell lines. The existence of antibodies specific for other nucleoside modifications makes this a system that is potentially applicable to a variety of deficiencies in the modification of both tRNA and rRNA.     

Translocation of a tRNA with an extended anticodon through the ribosome

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.     

Comparison of dissimilarity patterns of E coli, yeast and mammalian tRNAs.

A number of experimental approaches have been developed for identification of recognition (identity) sites in tRNAs. Along with them a theoretical methodology has been proposed by McClain et al that is based on concomitant analysis of all tRNA sequences from a given species. This approach allows an evaluation of nucleotide combinations present in isoacceptor tRNAs specific for the given amino acid, and not present in equivalent positions in cloverleaf structure in other tRNAs of the same organism. These elements predicted from computer analysis of the databank could be tested experimentally for their participation in forming recognition sites. The correlation between theoretical predictions and experimental data appeared promising. The aim of the present work consisted of introducing further improvements into McClain's procedure by: i), introducing into analysis a variable region in tRNAs which had not been previously considered; to accomplish this, 'normalization' of variable nucleotides was suggested, based on primary and tertiary structures of tRNAs; ii), developing a new procedure for comparison of patterns for synonymous and non-synonymous tRNAs from different organisms; iii), analysis of 3- and 4-positional contacts between tRNAs and enzymes in addition to a formerly used 2-positional model. A systematic application of McClain's procedure to mammalian, yeast and E coli tRNAs led to the following results: i), imitancy patterns for non-synonymous tRNAs of any amino acid specificity and from any organisms analysed so far overlap by no more than 30%, providing a structural basis for discrimination with high fidelity between cognate and non-cognate tRNAs; ii), the predicted identity sites are non-randomly distributed within tRNA molecules; the dominant role is ascribed to only two regions--anticodon and amino acid stem which are located far apart from one another at extremes of all tRNA molecules; iii), the imitancy patterns for synonymous tRNAs in lower (yeast) and higher (mammalian) eukaryotes are similar but not identical; iv), distribution of predicted identity sites in the cloverleaf structure in prokaryotes and eukaryotes is essentially different: in eubacterial tRNAs the major role in recognition plays anticodon and/or amino acid acceptor stem, whereas in eukaryotic (both unicellular and multicellular) tRNAs the remaining part of the molecules is also involved in recognition; v), the imitancy patterns of synonymous tRNAs from prokaryotes and eukaryotes are dissimilar, this observation leads to the prediction that the tRNA identity sites for the same amino acid in prokaryotes and eukaryotes may differ.