Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.     

Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA

tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.     

Physiological and biochemical studies on the function of 5-methyluridine in the transfer ribonucleic acid of Escherichia coli.

Matched pairs of transductant strains differing by the presence of absence of 5-methyluridine (ribothymidine) (m5U) in their transfer ribonucleic acid (tRNA) were used to study the function of this modified nucleoside in Escherichia coli. Ordinary measurements of growth rate in different media revealed no effect of the loss of m5U in tRNA. A gene located close to trmA (the structural cistron for the methyltransferase that produces m5U in tRNA), however, was found to reduce the growth rates significantly, depending on the medium and the temperature of cultivation. Measurement of codon recognition, macromolecular composition, tRNA binding to the ribosome, and the rate of protein chain elongation in vivo indicated no disadvantage caused by the lack of m5U. The regulation of ilv and his operons seemed also to be unaffected by the absence of m5U in the tRNA. In a mixed population experiment, however, cells possessing m5U in their tRNA seemed to have a distinct advantage over cells lacking this modified nucleoside. This experiment provides the first indication of the overall value of m5U in tRNA.     

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.     

Transductional mapping of gene trmA responsible for the production of 5-methyluridine in transfer ribonucleic acid of Escherichia coli.

The gene trmA, responsible for the production of 5-methyluridine (ribothymidine) in transfer ribonucleic acid, has been located at 79 min on the chromosomal map of Escherichia coli K-12. In five-factor crosses the gene order was shown to be argH-trmA-rif-thiA-metA. The co-transduction frequency between argH and trmA was 65%. Furthermore, the trmA5 mutation was shown to be recessive, in agreement with the notion that the trmA gene is the structural gene for the transfer tibonucleic acid (5-methyluridine) methyltransferase.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli.

This paper describes the regulation of a transfer ribonucleic acid (tRNA) biosynthetic enzyme, the tRNA(m5U)methyltransferase (EC 2.1.1.35). This enzyme catalyzes the formation of 5-methyluridine (m5U, ribothymidine) in all tRNA chains of Escherichia coli. Partial deprivation of charged tRNAVal can be imposed by shifting strains carrying a temperature-sensitive valyl-tRNA ligase from a permissive to a semipermissive temperature. By using two such strains differing only in the allelic state of the relA gene, it was possible to show the tRNA(m5U)methyltransferase to be stringently regulated. Upon partial deprivation of charged tRNAVal, the differential rate of tRNA(m5U)methyltransferase synthesis was found to decrease in a strain with stringent RNA control (relA+), whereas it increased in the strain carrying the relA allele. This increase of accumulation of tRNA(m5U)methyltransferase activity required protein synthesis. Thus, when tRNA is partially uncharged in the cell, the relA gene product influences the expression of tRNA(m5U)methyltransferase gene.     

Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.     

Cloning and expression of Thiobacillus ferrooxidans mercury ion resistance genes in Escherichia coli.

A search of various domestic isolates of Thiobacillus ferrooxidans revealed that some were fairly resistant to mercury ion. A proportion of mercury-resistant clones were able to volatilize mercury, and their corresponding gene was localized not in the plasmid DNA but in chromosomal DNA. This mercury ion resistance gene was cloned in Escherichia coli. E. coli carrying the recombinant plasmid was able to grow in the presence of more than 40 micrograms of HgCl2 per ml. Deletion analysis of the recombinant plasmid showed that the entire coding sequence of the mercury ion resistance gene was located within a 2.3-kilobase fragment of the chromosomal DNA from strain E-15. At least two polypeptides (molecular mass, 56 and 16 kDa, respectively) were coded by this fragment.     

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.     

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.     

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.     

Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli.

A 3.4-kilobase EcoRI restriction endonuclease fragment has been cloned from the facultatively photoheterotrophic bacterium Rhodobacter sphaeroides and shown to contain the structural gene (prkA) for phosphoribulokinase (PRK) activity. The PRK activity was characterized in Escherichia coli, and the product of the reaction was identified. The prkA gene was localized to a 1,565-base-pair EcoRI-PstI restriction endonuclease fragment and gave rise to a 33-kilodalton polypeptide both in vivo and in vitro. The gene product produced in E. coli was shown to be identical to the gene product produced in R. sphaeroides. The amino acid sequence for the amino-terminal region deduced from the DNA sequence confirmed that derived for partially purified PRK derived from both E. coli and R. sphaeroides. In addition, the 3.4-kilobase EcoRI restriction endonuclease fragment coded for a 37-kilodalton polypeptide of unknown function, and preliminary evidence indicates that this DNA fragment is linked to genes coding for other activities significant in photosynthetic carbon assimilation. The genetic organization and proposed operon structure of this DNA fragment are discussed.     

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.