Schizosaccharomyces pombe contains separate CC- and A-adding tRNA nucleotidyltransferases

A specific cytidine-cytidine-adenosine (CCA) sequence is required at the 3′-terminus of all functional tRNAs. This sequence is added during tRNA maturation or repair by tRNA nucleotidyltransferase enzymes. While most eukaryotes have a single enzyme responsible for CCA addition, some bacteria have separate CC- and A-adding activities. The fungus, Schizosaccharomyces pombe, has two genes (cca1 and cca2) that are thought, based on predicted amino acid sequences, to encode tRNA nucleotidyltransferases. Here, we show that both genes together are required to complement a Saccharomyces cerevisiae strain bearing a null mutation in the single gene encoding its tRNA nucleotidyltransferase. Using enzyme assays we show further that the purified S. pombe cca1 gene product specifically adds two cytidine residues to a tRNA substrate lacking this sequence while the cca2 gene product specifically adds the terminal adenosine residue thereby completing the CCA sequence. These data indicate that S. pombe represents the first eukaryote known to have separate CC- and A-adding activities for tRNA maturation and repair. In addition, we propose that a novel structural change in a tRNA nucleotidyltransferase is responsible for defining a CC-adding enzyme.     

Reactions at the 3' terminus of transfer ribonucleic acid. IX. tRNA nucleotidyltransferase activity in bacteriophage T4-infected Escherichia coli

There was no detectable increase in tRNA nucleotidyltransferase activity upon infection of $\text{Escherichia coli}$ A19 with bacteriophage T4. Three mutant strains which contained low levels of tRNA nucleotidyltransferase activity also showed no increase in activity after infection. tRNA nucleotidyltransferase was purified from both uninfected and T4-infected cells and examined for possible modification. It was found that enzyme purified from both types of cells eluted from DEAE cellulose at the same specific conductivity. In addition, the molecular weight of tRNA nucleotidyltransferase purified from both uninfected and T4-infected cells was approximately 45,000 daltons as determined by chromatography on Sephadex G-100. These results suggest that T4-infection does not lead to synthesis of a new virus-specific tRNA nucleotidyltransferase nor does it cause modification of the host enzyme.     

Purification of a hybrid molecule composed of tyrosine transfer RNA and its complementary DNA sequence

The transducing bacteriophage φ80psu_III⁺ carries one structural Escherichia coli gene specifying tyrosine tRNA.The r strand of bacteriophage φ80psu_III⁺ was hybridized with E. coli transfer RNA and the hybrid digested with Neurospora crassa endonuclease. The analysis of the products of enzymic digestion demonstrated the release of a cistron-hybrid composed of tyrosine tRNA and its complementary DNA sequence. The cistron-hybrid was purified from unhybridized DNA by cesium sulphate density-gradient centrifugation and gel filtration.The ratio between tyrosine tRNA and its complementary DNA sequence in the final product was 1:1 as demonstrated by radioisotopic analysis. This purification represents a 30,000-fold enrichment of the E. coli genome for a specific DNA sequence.     

The transfer RNA processing defect in Escherichia coli strains BN and CAN is not due to a mutation in RNAase D or RNAase II

Escherichia coli strains BN and CAN are unable to support the growth of bacteriophage T4 psu₁⁺-amber double mutants. For strain BN, this phenotype has been attributed to a defect in 3′ processing of the precursor to psu₁⁺ tRNA^Ser. Since RNAase D and RNAase II are the only well-characterized 3′ exoribonucleases to be implicated in tRNA processing, the status of these activities and their genes in the mutant strains was investigated. Although extracts of strains BN and CAN were defective for hydrolysis of the artificial tRNA precursor, tRNA-C-U, these strains contained normal levels of RNAase D and RNAase II, and purified RNAase D or RNAase II could only partially complement the mutant extracts. Introduction of the wild-type RNAase D gene into strains BN and CAN did not correct the mutant phenotype. Likewise, strains defective in RNAase D and/or RNAase II plated T4psu₁⁺-amber phage normally. These results indicate that the tRNA processing defect in strains BN and CAN is not due to a mutation in either RNAase U or RNAase II. The possibility that the mutation in these strains affects another exoribonuclease or a factor influencing the activity and specificity of RNAase D or RNAase II is discussed.     

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su⁺ 3 tRNA₁^Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.     

The Bacillus subtilis Nucleotidyltransferase Is a tRNA CCA-Adding Enzyme

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3′ poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the β and γ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca, following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3′ polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.     

Codon recognition by glycine transfer RNAs of Escherichia coli in vivo

In order to provide evidence concerning the nature of codon recognition by isoaccepting transfer RNAs of Escherichia coli in vivo, we have carried out genetic experiments with appropriate glycine tRNA mutants that display altered coding specificities. Others have demonstrated that in the ribosome-triplet binding assay glyT tRNA is the only glycine-accepting tRNA that can respond to the glycine codon GGA. Using glyT-derived translational suppressors that respond to AGA, GAA, AAG, UGA and UGG, we have shown that glyT tRNA is indeed the only GGA-reader in vivo and that, rather than using a “two out of three” method of codon recognition, glycine tRNAs in the E. coli cell recognize all three nucleotides of a codon. Furthermore, the data suggest that some mutationally altered glyT tRNAs exhibit an unorthodox wobble in response to the first or second position of a codon.     

Gross map location of Escherichia coli transfer RNA genes.

Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F^? strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNA_f1^Met gene newly determined, the λ-transducing phage carrying the tRNA_f1^Met gene was found.     

Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA₁^leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA₁^met and su⁺_III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

Identification of transfer RNA suppressors in Escherichia coli. II. Duplicate genes for tRNA2Gln.

The number of gene copies for tRNA₂^Gln in λpsu⁺2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su⁺2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNA_m. When the su⁺2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA₂^Gln, instead of the amber-suppressing tRNA₂^Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA₂^Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA₂^Gln genes, one su⁺2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNA_m^Met gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA₂^Gln at 15 minutes on the bacterial chromosome. su⁺2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA₂^Gln genes could also become ochre suppressors. A tRNA_m^Met gene is located near, but not between, these two tRNA₂^Gln genes.     

The absence of ribothymidine in specific eukaryotic transfer RNAs. I. Glycine and threonine tRNAs of wheat embryo

Ribothymidine, generally considered a universal nucleotide in tRNA, is completely absent in five specific wheat embryo tRNAs. These consist of two species of glycine tRNA and three species of threonine tRNA. These tRNAs, all extensively purified, are acceptable substrates for $\text{E. coli}$ - ribothymidine forming-uracil methylase, which produces one mole of ribothymidine per mole of tRNA. These five tRNAs account for about 90% of the wheat embryo tRNAs which are substrates for this methylase. Nucleotide sequence analysis of one of these tRNAs, tRNA^Gly_I, confirmed both the complete absence of ribothymidine at position 23 from the 3′end, and the presence of uridine at that site instead. In addition, it is shown that methylation with $\text{E. coli}$ uracil methylase quantitatively converts uridine at position 23 to ribothymidine, while no other uridine in the molecule is affected.Using $\text{E. coli}$ uracil methylase as an assay we have detected this class of ribothymidine lacking tRNA, in each case consisting of a few specific species, in other higher organisms, such as wheat seedling, fetal calf liver and beef liver, in addition to wheat embryo. We could not detect this class of tRNA in $\text{E. coli}$ or yeast tRNA.     

Nucleotide sequence determination of bacteriophage T4 glycine transfer ribonucleic acid

The nucleotide sequence of a T4 tRNA with an anticodon for glycine has been determined using ³²P-labeled material from T4-infected cultures of Escherichia coli. The sequence is: pGCGGAUAUCGUAUAAUGmGDAUUACCUCAGACUUCCAAψCUGAUGAUGUGAGTψCGAUUCUCAUUAUCCGCUCCA-OH. The 74 nucleotide sequence can be arranged in the classic cloverleaf pattern for tRNAs. The anticodon of T4 tRNA^Gly is UCC with a possible modification of the U. The tRNA molecule would thus be expected to recognize the glycine codons GGG and GGA. Comparative analysis of tRNAs^Gly from T2 and T6 indicate that their sequences are identical with that from T4.     

Translation of the UGA triplet in vitro by tryptophan transfer RNA's

Tryptophan transfer RNA from the UGA-suppressing strain of Escherichia coli CAJ64 was purified and assayed for suppressor activity in vitro in two ways: by translation of the bacteriophage T4 lysozyme messenger RNA bearing a UGA mutation, and by translation of poly(U-G-A). Purified tRNA^Trp, and no other fraction, stimulates lysozyme synthesis 30-fold above the level seen when comparable amounts of tryptophan tRNA from the non-suppressing strain, CA244, were added; it also translates poly(U-G-A) as polytryptophan more efficiently than the su⁻ tRNA. Tryptophan tRNA from the non-suppressing strain is active in the assays but far less so than CAJ64 tRNA^Trp, and this is consistent with the leakiness of su⁻ strains. Since the nucleotide sequences of these tryptophan tRNA's are known (Hirsh, 1971), it is concluded that tRNA with a CCA anticodon recognizes the UGA triplet and this recognition is improved by a nucleotide change elsewhere in the molecule.     

A role for ribonuclease III in synthesis of bacteriophage T4 transfer RNAs.

Synthesis of T4 tRNA^Gln depends on normal levels of $\text{Escherichia}\text{coli}$ ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNA^Gln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNA^Gln points to an essential cleavage by the enzyme of a precursor RNA containing tRNA^Gln.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.