Purification of potential 3'' processing nucleases using synthetic tRNA precursors.

The synthetic tRNA precursors, tRNA-C-114C]U and tRNA-C-C-A-[14C]C-C, as well as poly (a) and diesterase-treated tRNA, have been used to identify and purify potential 3'processing nucleases. Four activities have been separated by this analysis; and three of them have been characterized. Two of the enzymes, which are well-separated on hydroxylapatite columns, act on poly(A), require K+ and Mg2+ for activity, and have molecular weights of about 90,000. These activities have properties previously ascribed to RNase II. The third enzyme does not act on poly(A), requires Mg2+ for activity, and has a molecular weight of about 60,000. It is identical to RNase D, previously characterized as an exonuclease acting on tRNAs with altered structure. Each of the enzymes can remove nucleotides from the tRNA precursor containing extra nucleotides beyond the 3'terminus, whereas they are relatively inactive with intact tRNA or tRNA-C-U. The greatest specificity was displayed by RNase D. The possibility that RNase D is a 3'processing nuclease is discussed.     

Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing.

A survey of RNases in Xenopus laevis oocytes has been carried out to identify potential tRNA-processing enzymes in this system. Using a variety of specific and nonspecific substrates, we have shown that oocytes contain multiple RNases with various specificities. Three activities that could cleave the extraneous residues from the artificial tRNA precursor, tRNA-C-[14C]U-C, to generate a substrate for -C-C-A addition by tRNA nucleotidyltransferase were identified. One of these was a cytoplasmic exonuclease which generated predominantly tRNA-C, whereas the other two were nuclear endonucleases which cleaved the precursor to generate tRNA-N. The possible involvement of these activities in 3' tRNA processing in oocytes is discussed.     

Detection of nucleoside Q precursor in methyl-deficient E.coli tRNA.

32P-Labeled tRNAAsn was isolated from methyl-deficient E. coli tRNA. Nucleotide sequence analysis showed that tRNAAsn contains three derivatives of the Q nucleoside, possibly Q precursors, in addition to guanosine in the first position of the anticodon. One of the Q precursors was isolated on a large scale. Its UV spectra were identical with those of normal Q, indicating that 7-deazaguanosine structure having a side chain at position C-7 is complete in the Q precursor. No radioactivity was incorporated into Q or Q precursors from either [methyl-14C]methionine, [1-14C]methionine or [U-14C]methionine, showing that methionine was not directly involved in the formation of Q.     

Utilization of an Escherichia coli mutant for carbon-13 enrichment of tRNA for NMR studies.

The enrichment of tRNA at specific sites with carbon-13 has been accomplished in vivo using a mutant of Escherichia coli. A relaxed strain of E. coli auxotrophic for methionine was grown in a specifically defined medium supplemented with either [14C] or [13C]-methyl labeled methionine. Cells were collected at the end of the log-phase of growth and tRNA was extracted. Analysis of the radioactivity of the [14C]-labeled tRNA established an incorporation ratio of three labeled carbons per tRNA molecule. Incorporation of the [14C]-label in vivo was confined to the methylation of nucleotides as determined by thin layer chromatography of nucleotides resulting from a ribonuclease digestion of [14C]-labeled tRNA. The carbon-13 NMR spectrum of [13C]-enriched tRNA indicated a similar degree of incorporation into the methylated nucleotides by the substantial enhancement of [13C]-methyl NMR signals only. Assignment of signals has been made for the methyl groups of ribothymidine and N7-methylguanosine in E. coli tRNA.     

Stable tRNA precursors in HeLa cells.

Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors.     

tRNA nucleotidyltransferase is not essential for Escherichia coli viability.

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.     

Limited hydrolysis of tRNA by phosphodiesterase.

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.     

Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec

Eukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes.     

Half-life of tRNA Containing N6(Δ2-isopentenyl)adenosine in Lactobacillus acidophilus ATCC 4963

tRNA containing N⁶-(Δ²-isopentenyl)adenosine may be precursors for the plant hormone cytokinin. To discriminate between tRNA containing and not containing cytokinin nucleotides, double labelling experiments were made by the use of [2¹⁴C]-mevalonic acid and [³H-methyl]-methionine. At a generation cycle of 2 h for Lactobacillus acidophilus ATCC 4963, the half-lives of tRNA labelled with [³H-methyl]-methionine and [2-¹⁴C]-mevalonic acid are similar, namely 3 h. Isopentenylation of tRNA could be measured to be maximally 1:10.     

Asymmetric maturation of a dimeric transfer RNA precursor

Six of the eight transfer RNAs coded by bacteriophage T4 are synthesized via three dimeric precursor molecules. The sequences of two of these have been determined. Both of these precursors give rise to equimolar amounts of the cognate tRNA molecules in vivo. In contrast, even in wild-type infections, tRNA^Ile is present in ≤ 30% the amount of tRNA^Thr, with which it is processed from a common dimeric precursor.We have now determined the sequence of this dimer. In addition to the nucleotides present in tRNA^Thr and tRNA^Ile, it contains nine precursor-specific residues, located at the 5′ and 3′ termini and at the interstitial junction of the two tRNA sequences. While the three dimers share the majority of structural features in common, pre-tRNA^{Thr + Ile} is the only case in which an encoded tRNA 3′ -C-C-A terminus is present in the interstitial region.The processing of this dimer in various biosynthetic mutants has been analyzed in vivo and in vitro and shown to be anomalous in several respects. These results suggest that the apparent underproduction of tRNA^Ile can be explained by a novel processing pathway that generates a metabolically unstable tRNA^Ile product. Data from DNA sequence analysis of the T4 tRNA gene cluster (Fukada & Abelson, 1980) support the conclusion that the asymmetric maturation of this precursor is a consequence of the unique disposition of the -C-C-A sequence. These results argue that gene expression can be modulated at the level of RNA processing. The biological significance of this phenomenon is discussed in relation to evidence that tRNA^Ile has a unique physiological role.     

Sequence analysis of 5''[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis.

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.     

Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus

RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution. RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.     

Escherichia coli tyrosyl- and methionyl-tRNA synthetases display sequence similarity at the binding site for the 3'-end of tRNA

Covalent modification of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) by the 2',3'-dialdehyde derivative of tRNATyr (tRNAox) resulted in a time-dependent inactivation of both ATP-PPi exchange and tRNA aminoacylation activities of the enzyme. In parallel with the inactivation, covalent incorporation of approximately 1 mol of [14C]tRNATyrox/mol of the dimeric synthetase occurred. Intact tRNATyr protected the enzyme against inactivation by the tRNA dialdehyde. Treatment of the TyrRS-[14C]tRNATyr covalent complex with alpha-chymotrypsin produced two labeled peptides (A and B) that were isolated and identified by sequence analysis. Peptides A and B are adjacent and together span residues 227-244 in the primary structure of the enzyme. The three lysine residues in this sequence (lysines-229, -234, and -237) are labeled in a mutually exclusive fashion, with lysine-234 being the most reactive. By analogy with the known three-dimensional structure of the homologous tyrosyl-tRNA synthetase from Bacillus stearothermophilus, these lysines should be part of the C-terminal domain which is presumed to bind the cognate tRNA. Interestingly, the labeled TyrRS structure showed significant similarities to the structure around the lysine residue of E. coli methionyl-tRNA synthetase which is the most reactive toward tRNAMetf(ox) (lysine-335) [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180].     

The nucleotide sequence of a methionine tRNA which functions in protein elongation in mouse myeloma cells.

The major form of methionine tRNA operational in the elongation of protein synthesis in mouse myeloma cells was purufied from these cells after they had been cultured in the presence of [32P]-phosphate. This [32P]tRNA4-Met species was then digested with T1 RNase or pancreatic RNase so as to obtain both complete and partial RNase digestion products. The nucleotide sequences of these fragments were analysed to enable the derivation of the complete primary structure of this tRNA. tRNA4-Met of mouse myeloma cells is 76 nucleotides in length and contains 15 modified nucleotides. It is the only tRNA yet sequenced which has been found to possess the minor nucleoside 2-methylguanosine (m2G) within the amino acid (a) stem, and also to have an anticodon (c) stem of only 4 and not 5 base-pairs. The loop IV sequence of eukaryotic initiator methionine tRNA (tRNAf-Met) species, -A-U-C-G-m1A-A-A-, IS NOT FOUND IN TRNA4-Met and is therefore absent from at least one of the methionine tRNAs functioning in polypeptide elongation in mammalian cells. This is consistent with the suggested importance of this loop structure in the initiator function of tRNAf-Met in eukaryotic organisms. Three distinct regions of the tRNA cloverleaf, the (b) stem, the anticodon loop (loop II), and loop III, are substantially conserved in structure between tRNAf-Met and tRNA4-Met of mouse myeloma cells. These regions of the structures of mammalian methionine tRNAs probably do not determine whether a certain tRNA-Met will function in the initiation or elongation of protein synthesis, although they might be important in tRNA-Met recognition if the different cytoplasmic tRNA-Met species of mammalian cells are aminoacylated by a single activating enzyme.     

Primary structure of tRNA Thr 1a and b from brewer's yeast]

One of the two major species of brewer's yeast tRNA threonine (tRNA Thr 1) has been purified by countercurrent distribution followed by two chromatographic steps (respectively on a Sepharose 4B and a BD-cellulose column). Complete digestion with pancreatic and T1 RNases and a partial hydrolysis with T1 RNase followed by the isolation and determination of the nucleotide sequences of the resulting fragments permitted the derivation of its primary structure. tRNA Thr 1 is in fact a mixture of two subspecies differing only by a A49-U65 base pair in 50 per cent of the molecules which is replaced by a G49-C65 pair in the other 50 per cent. These two subspecies consist of 76 nucleotide residues including 14 minor nucleotides. They show a characteristic m3C at the 3'terminal end of the anticodon loop, an anticodon I-G-U followed by t6A and C48, uncompletely modified (50 per cent) to m5C within the 5 nucleotides long extra-arm. The minor nucleotides m2G m2 2G are located at positions in which they generally occur in the tRNA structures as does m1A within the T-psi-C loop.     

1,7-Diethylguanosine formation in tRNA chemical ethylation by ethionine and ethylnitrosourea

The mechanism of ethionine carcinogenesis and more generally the relationship between alkylation of nucleic acids by chemical carcinogens and oncogenesis still remain obscure. In the present study the rat liver tRNA ethylation by L-[ethyl-1-3H]ethionine was reinvestigated by examining in particular the highly radioactive 'pyrimidine-nucleotide-like' fraction found earlier in acid hydrolysates of hepatic tRNA from ethionine-treated rats. The following results were obtained: (1) ultraviolet-spectral and chromatographic analyses showed the presence of 1,7-diethylguanosine in this 'pyrimidine-nucleotide-like' fraction; (2) the dialkyl compound was recovered exclusively in the form of imidazole-ring-opened derivatives. When [1-14C]ethylnitrosourea was used as alkylating agent, the in vivo ethylation pattern of tRNA from various organs of rat showed an analogous radioactive 'pyrimidine-nucleotide-like' fraction as main radioactive product. On the contrary, tRNA ethylation pattern after in vitro reaction with [1-14C]ethylnitrosourea exhibited a main radioactivity peak (85% of the total radioactivity recovered) in coincidence of the chromatographic area of 1,7-diethylguanine. The 1,7-diethylguanosine moieties of tRNA were extremely labile both under physiological and alkaline conditions. The 1,7-diethylguanine-associated radioactivity was completely lost from [14C]ethyl-tRNA after only 7 h incubation at 37 degrees C and pH 7.3, while at pH 11.4 this process was preceded by the conversion of the 1,7-diethylguanosine residues into imidazole-ring-opened derivatives.     

tRNA Creation by Hairpin Duplication

Many studies have suggested that the modern cloverleaf structure of tRNA may have arisen through duplication of a primordial hairpin, but the timing of this duplication event has been unclear. Here we measure the level of sequence identity between the two halves of each of a large sample of tRNAs and compare this level to that of chimeric tRNAs constructed either within or between groups defined by phylogeny and/or specificity. We find that actual tRNAs have significantly more matches between the two halves than do random sequences that can form the tRNA structure, but there is no difference in the average level of matching between the two halves of an individual tRNA and the average level of matching between the two halves of the chimeric tRNAs in any of the sets we constructed. These results support the hypothesis that the modern tRNA cloverleaf arose from a single hairpin duplication prior to the divergence of modern tRNA specificities and the three domains of life. [Reviewing Editor: Dr. Niles Lehman]     

Number of genes and base composition of mitochondrial tRNA from Saccharomyces cerevisiae.

Increasing amounts of mitochondrial [32P] tRNA (4S fraction), were hybridized with mitochondrial DNA OF Saccharomyces cerevisiae. At saturation, the calculated number of genes for 4S mitochondrial RNA was 20. Mitochondrial [32P] tRNA eluted from the hydrids obtained either with an excess of tRNA or an excess of DNA showed, after alkaline hydrolysis and chromatography, a G+C content of 28 and 35 p. cent respectively. This last value is similar to that found with the total 4S fraction. The odd nucleotides T (about 1T per sequence), U, hU are present in mitochondrial tRNA. Some sequence may begin with pG.