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.     

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.     

Processing of RNA in Escherichia coli is limited in the absence of ribonuclease III,ribonuclease E and ribonuclease P

A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with ³²P_i at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P.     

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.     

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.     

Increased Inactivation of Messengers in <Emphasis Type="Italic">Escherichia coli ts</Emphasis> Mutant

WE recently described some of the properties of a temperature sensitive mutant of Escherichia coli (refs. 1–3 and unpublished work) in which RNAase II activity is increased on transfer to the non-permissive temperature^1,2, while the functional half-life of β-galactosidase mRNA¹ and the chemical half-life of the lac Operon mRNA³ are decreased. Questions raised by these studies were (a) can the strain be considered a general messenger RNAase mutant and (b) what is the direction of messenger inactivation in this strain? The latter question is particularly interesting since the increased RNAase activity in this strain is that of RNAase II (unpublished work) which degrades RNA molecules in the 3′ to 5′ direction⁴, while mRNA is known to be degraded in the 5′ to 3′ direction^5,6.     

Cleavage of tRNA precursors by the RNA subunit of E. coli ribonuclease P (M1 RNA) is influenced by 3''-proximal CCA in the substrates

tRNA precursor molecules that contain the CCA sequence found at the 3' termini of all mature tRNAs are cleaved in vitro more readily by M1 RNA, the catalytic subunit of E. coli RNAase P, than precursors that lack this sequence. The sensitivity to the CCA sequence is not apparent when precursors are cleaved by the reconstituted RNAase P holoenzyme that contains both M1 RNA and the protein subunit. These results have been obtained with monomeric precursor molecules encoded by the E. coli and human chromosomes and with three dimeric precursor molecules encoded by the bacteriophage T4 genome. The data are in agreement with previous results concerning T4 tRNA biosynthesis in vivo and show that the CCA sequence is important for the processing of precursors to tRNAs.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

In vitro processing of E. coli tRNA precursors

Using E. coli tRNA precursors isolated from an RNAase P mutant strain, we have studied the steps required for the formation of tRNAs having a mature primary sequence in vitro. Our results suggest that at least three different enzymatic activities can participate in the processing of tRNA precursors.     

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.     

Oxidation of 4-thiouridine in intact Escherichia coli tRNAs.

Treatment of intact tRNAs from Escherichia coli B with mild oxidizing agents, such as KI-I₂, appears to quantitatively oxidize the 4-thiouridine present in these molecules to the disulfide form as judged by the loss of absorbance near 330 nm. Chromatography of these oxidized tRNAs on Sephadex G-75 did not reveal tRNA dimers or larger aggregates, suggesting intra- rather than intermolecular disulfide-bond formation. Enzymatic hydrolyses of both unlabeled and ³⁵S-labeled oxidized tRNAs followed by chromatography on columns of Sephadex G-25 indicated that 4-thiouridine did form covalent linkages with some component(s) in the tRNA that were reversible upon reduction. It was not clear whether 4-thiouridine formed disulfides only with itself, other sulfurcontaining nucleosides, or some non-sulfur-containing component. Data presented suggest that an earlier report on the isolation of 4-thiouridylate disulfide from oxidized tRNAs of E. coli was an artifact, resulting from oxidation of the thionucleotide during chromatography on Bio-Gel.     

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNA^Met_f has been further characterized. This substituent is responsible for the ¹⁹F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution ¹⁹F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield ¹⁹F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNA^Met_m and tRNA^Val_l, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with ¹⁹F spectrum of the model compound 5′-deoxy-5-fluoro-5,6-dihydrouridine (dH⁵₆FUrd) (δ_FUra = ? 31.4 ppm; J_HF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH⁵₆FUrd is considerably more alkali labile than 5,6-dihydrouridine (H⁵₆Urd). At pH 8.9, where H⁵₆Urd is stable, dH⁵₆FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a ¹⁹F resonance at ? 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The ¹⁹F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the ¹⁹F signal at ? 15 ppm. Hydrolysis of this tRNA with RNAase T₂ produces a sharp doublet 33 ppm upfield (J_HF = 45 Hz). Similarities of the ¹⁹F chemical shift and coupling constant to those of dH⁵₆FUrd, allows assignment of the peak at ? 33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.     

Relationship between the structure and function of Escherichia coli initiator tRNA

Through functional studies of mutant tRNAs, we have identified sequence and/or structural features important for specifying the many distinctive properties of E coli initiator tRNA. Many of the mutant tRNAs contain an anticodon sequence change from CAU→CUA and are now substrates for E coli glutaminyl-tRNA synthetase (GlnRS). We describe here the effect of further mutating the discriminator base 73 and nucleotide 72 at the end of the acceptor stem on: i) recognition of the mutant tRNAs by E coli GlnRS; ii) recognition by E coli methionyl-tRNA transformylase; and iii) activity of the mutant tRNAs in initiation in E coli. For GlnRS recognition, our results are, in general, consistent with interactions found in the crystal structure of the E coli GlnRS-glutamine tRNA complex. The results also support our previous conclusion that formylation of initiator tRNA is important for its function in initiation.     

A tRNA's fate is decided at its 3′ end: Collaborative actions of CCA-adding enzyme and RNases involved in tRNA processing and degradation

tRNAs are key players in translation and are additionally involved in a wide range of distinct cellular processes. The vital importance of tRNAs becomes evident in numerous diseases that are linked to defective tRNA molecules. It is therefore not surprising that the structural intactness of tRNAs is continuously scrutinized and defective tRNAs are eliminated. In this process, erroneous tRNAs are tagged with single-stranded RNA sequences that are recognized by degrading exonucleases. Recent discoveries have revealed that the CCA-adding enzyme – actually responsible for the de novo synthesis of the 3′-CCA end – plays an indispensable role in tRNA quality control by incorporating a second CCA triplet that is recognized as a degradation tag. In this review, we give an update on the latest findings regarding tRNA quality control that turns out to represent an interplay of the CCA-adding enzyme and RNases involved in tRNA degradation and maturation. In particular, the RNase-induced turnover of the CCA end is now recognized as a trigger for the CCA-adding enzyme to repeatedly scrutinize the structural intactness of a tRNA. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Apparent non-involvement of transfer RNA nucleotidyltransferase in the biosynthesis of Escherichia coli suppressor transfer RNAs

Transfer RNA nucleotidyltransferase has previously been shown to be required for the normal growth of Escherichia coli and for the biosynthesis of some bacteriophage T4 tRNAs. In order to obtain information about the involvement of this enzyme in E. coli tRNA biosynthesis we have measured the level of activity of suppressors 1 to 6 in strains carrying either a cca⁺ or cca allele. We found that cca strains, deficient in tRNA nucleotidyltransferase, contained the same amount of suppressor activities as the wild-type cca⁺ strains as determined by suppression of nonsense mutations in both E. coli alkaline phosphatase and in genes of bacteriophage T4. The results suggest that tRNA nucleotidyltransferase is not required for the biosynthesis of tRNAs specified by suppressors 1 to 6.     

Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs

There exists a similarity among the synonymous codon choice patterns of the yeast nuclear genes that have been sequenced thus far although these genes encode different types of protein molecules, and the patterns are significantly different from those of Escherichia coli genes. Based on constraints caused by the availability of E. coli transfer RNAs and the nature of their codon recognition related to the modified nucleotides at the anticodon wobble position, the characteristic patterns of synonymous codon choice commonly found for E. coli genes have been almost completely explained (Ikemura, 1981a, b). In the present paper, tRNAs of the yeast Saccharomyces cerevisiae were separated by two-dimensional polyacrylamide gel electrophoresis and the relative abundance of purified tRNA molecules was measured on the basis of molecular numbers in cells. A strong correlation between tRNA abundance and codon choice was found for each nuclear gene of yeast, but the correlation was less significant for 2μ plasmid genes. According to the criteria proposed for E. coli genes (Ikemura, 1981b) the order of codon preference in yeast nuclear genes was predicted based on the abundance of yeast isoaccepting tRNAs and on the nature of the modified nucleotides at their anticodons. Clear correlations between predictions and the actual preferences among synonymous codons were revealed, indicating that the codon choices in yeast genes are also constrained by a combination of tRNA availability and nature of its codon recognition. Then the difference in synonymous codon use between the two organisms can be attributed to the difference in these two factors.