Escherichia coli tRNA (uracil-5-)-methyltransferase: Inhibition by analogues of adenosylhomocysteine.

Structural analogues of adenosylhomocysteine (AdoHcy) have been tested as inhibitors of a tRNA(uracil-5-)-methyltransferase preparation obtained from Escherichia coli. All analogues tested gave linear competitive inhibition kinetics with adenosylmethionine (AdoMet) as the variable substrate. Comparison of the Ki values obtained leads to the following conclusions concerning the specificity of the AdoMet-AdoHcy binding site on the enzyme: (i) the terminal amino group of the amino acid moiety is necessary for activity; (ii) both a chiral change of the asymmetric carbon atom of homocysteine and the presence of the terminal carboxyl group contribute little towards inhibitory activity; (iii) analogues in which the amino function of the adenyl moiety is modified or substituted are still potent inhibitors; (iv) inhibitor specificity is considerably reduced when adenine is replaced by a pyrimidine base.     

Kinetic studies of Escherichia coli transfer RNA (uracil-5-)-methyltransferase.

The kinetic mechanism of a semipurified tRNA (uracil-5-)-methyltransferase (EC 2.1.1.35) preparation obtained from Escherichia coli has been studied at pH 9.0 in the presence and absence of products. The initial velocity and product inhibition patterns are consistent with a random order of addition of adenosylmethionine and transfer RNA to separate and independent binding sites on the enzyme. Values have been determined for the Michaelis and product inhibitor constants.     

Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12.

A hybrid plasmid from the Clarke and Carbon collection has been isolated. This plasmid carries the trmA gene of E. coli, which is necessary for the formation of 5-methyluridine (m5U,ribothymidine) present in all transfer ribonucleic acid (tRNA) chains of the organism so far sequenced. A restriction map of the argCBH-trmA regions is presented. By using cloning in vitro, the trmA gene was located on a 2.9-kilobase pair deoxyribonucleic acid (DNA) fragment. These results and comparison with lambda dargECBH transducing phages established the gene order: argECBH trmA bfe in the 88-min region of the E. coli chromosomal map. Plasmids carrying this 2.9-kilobase pair DNA fragment overproduce the enzyme tRNA(m5U)methyltransferase (EC 2.1.1.35) 20 to 40 times. When this 2.9-kilobase pair chromosomal DNA fragment was expressed in a minicell system, a polypeptide of a molecular weight of 42,000 was synthesized. This polypeptide was tentatively identified as the tRNA(m5U)methyltransferase. These results support the earlier suggestion that the trmA gene is the structural gene for the tRNA(m5U)methyltransferase.     

Genetic mapping and cloning of the gene (trmC) responsible for the synthesis of tRNA (mnm5s2U)methyltransferase in Escherichia coli K12

Summary The trmC gene, responsible for the formation of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U) from 2-thiouridine, present in the first position in the anticodon of some tRNAs, has been located at 50.5 min on the Escherichia coli K12 chromosome. Results from transductional mapping suggest that the trmC gene is located counter-clockwise of aroC. A ColE1 hybrid plasmid carrying the aroC ⁺, trmC ⁺ and hisT ⁺ genes was isolated, and the gene order was established, by subcloning, to be hisT-trmC-aroC. The trmC gene is located 1.9 kb from the aroC gene. Two mutations (trmC1 and trmC2) were shown to be recessive, suggesting that the trmC gene is the structural gene for the tRNA-(mnm⁵s²U)methyltransferase.     

Acquisition of a bacterial RumA-type tRNA(uracil-54, C5)-methyltransferase by Archaea through an ancient horizontal gene transfer

The Pyrococcus abyssi genome displays two genes possibly coding for S-adenosyl-l-methionine-dependent RNA(uracil, C5)-methyltransferases (PAB0719 and PAB0760). Their amino acid sequences are more closely related to Escherichia coli RumA catalysing the formation of 5-methyluridine (m(5)U)-1939 in 23S rRNA than to E. coli TrmA (tRNA methyltransferase A) methylating uridine-54 in tRNA. Comparative genomic and phylogenetic analyses show that homologues of PAB0719 and PAB0760 occur only in a few Archaea, these genes having been acquired via a single horizontal gene transfer from a bacterial donor to the common ancestor of Thermococcales and Nanoarchaea. This transfer event was followed by a duplication event in Thermococcales leading to two closely related genes. None of the gene products of the two P. abyssi paralogues catalyses in vitro the formation of m(5)U in a P. abyssi rRNA fragment homologous to the bacterial RumA substrate. Instead, PAB0719 enzyme (renamed (Pab)TrmU54) displays an identical specificity to TrmA, as it catalyses the in vitro formation of m(5)U-54 in tRNA. Thus, during evolution, at least one of the two P. abyssi RumA-type enzymes has changed of target specificity. This functional shift probably occurred in an ancestor of all Thermococcales. This study also provides new evidence in favour of a close relationship between Thermococcales and Nanoarchaea.     

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.     

The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity

The 5-methyluridine is invariably found at position 54 in the TPsiC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase ((Pab)TrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of (Pab)TrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 A resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of (Pab)TrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by (Pab)TrmU54. These results were used to model yeast tRNA(Asp) in the (Pab)TrmU54 structure to get further insights into the different RNA specificities of RumA and (Pab)TrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to (Pab)TrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TPsiC loop conformational change has to occur for the flipping of the target uridine into the (Pab)TrmU54 active site during catalysis.     

Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12

Summary The trmD gene, which governs the formation of 1-methyl-guanosine (m¹G) in transfer ribonucleic acid (tRNA), has been located by phage P1 transduction at 56 min on the chromosomal map of Escherichia coli. Cotransduction to tyrA at 56 min is 80%. From the Clarke and Carbon collection a ColE1-tyrA ⁺ hybrid plasmid was isolated, which carried the trmD ⁺ gene and was shown to over-produce the tRNA (m¹G)methyltransferase. By subcloning restriction enzyme fragments in vitro, the trmD ⁺ gene was located to a 3.4 kb DNA fragment 6.5 kb clockwise from the tyrA ⁺ gene. The mutation trmD1, which renders the tRNA (m¹G) methyltransferase temperaturesensitive both in vivo and in vitro could be complemented by trmD ⁺ plasmids. These results suggest that the gene trmD ⁺ is the structural gene for the tRNA (m¹G)methyltransferase (EC 2.1.1.3.1).     

Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-1 mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally. The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cls mutants depended on the dosage of the pss gene, and attempts to transfer a null allele of the cls gene into a pss-1 mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.     

Genetic mapping of pheU, an Escherichia coli gene for phenylalanine tRNA

We report the genetic mapping of pheU , an Escherichia coli gene for phenylalanine tRNA. This gene was located near 94.5 min on the E. coli map. There are no other known tRNA or ribosomal genes in its immediate vicinity.     

The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase

Fragments of Escherichia coli FUra-tRNA(1Val) as small as 15 nucleotides form covalent complexes with tRNA (m5U54)-methyltransferase (RUMT). The sequence essential for binding includes position 52 of the T-stem and the T-loop and extends toward the 3' acceptor end of FUra-tRNA. The in vitro synthesized 17mer T-arm of E. coli tRNA(1Val), composed of the seven-base T-loop and 5-base-pair stem, is a good substrate for RUMT. The Km is decreased 5-fold and kcat is decreased 2-fold compared to the entire tRNA. The T-arm structure could be further reduced to an 11mer containing the loop and two base pairs and still retain activity; the Km was similar to that of the 17mer T-arm, whereas kcat was decreased an additional 20-fold. The data indicate that the primary specificity determinants for the RUMT-tRNA interaction are contained within the primary and secondary structure of the T-arm of tRNA.     

Nucleotide sequence of the Escherichia coli tRNA(3Leu) gene

A 300-nucleotide sequence was determined which includes the tRNA(3Leu) coding region and the flanking sequences.     

1-(3-C-methyl-beta-D-ribofuranosyl)uracil-5'-phosphate--a terminator of RNA synthesis catalyzed by RNA polymerase from E. coli

It is shown that 1-(3'-C-methyl-beta-D-ribofuranosyl)uracil 5'-triphosphate is a terminator of RNA synthesis and may be used for nucleic acid sequencing with DNA-dependent RNA polymerase from E. coli.     

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.     

Affinity chromatography of Escherichia coli (m5U54)-methyltransferase on tRNA-agarose.

tRNA-agarose was prepared by condensing periodate-oxidized tRNA to an agarose matrix containing hydrazide functional groups. The tRNA-agarose was used to take partially purified tRNA (m5U54)-methyltransferase to homogeneity. The method is simple and reproducible and gives high yields.     

Temperature sensitive mutants of Escherichia coli for tRNA synthesis

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3⁺-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA^Tyr was demonstrated. Electrophoretic fractionation of ³²P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions.