Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I

The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.     

Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an intestinal anaerobic bacterium which possesses an inducible bile acid 7-dehydroxylation activity. Two cholic acid-induced polypeptides with apparent molecular weights of 27,000 and 45,000, respectively, coeluted with bile acid 7-dehydroxylation activity upon anaerobic high-performance gel filtration chromatography of crude cellular protein extracts. The 45,000-dalton polypeptide was purified to greater than 95% homogeneity by high-performance liquid chromatography gel filtration and high-performance liquid-DEAE chromatography. The first 28 amino acid residues of the N terminus of this polypeptide were determined by gas-phase sequencing, and a corresponding mixed oligonucleotide (20-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.6-kilobase fragment which hybridized to the 32P-labeled 20-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA and ligated into bacteriophage lambda gt11. Recombinant phage containing the putative gene encoding the 45,000-dalton polypeptide were detected with the 32P-labeled 20-mer by plaque hybridization techniques. The insert was 2.6 kilobases in length and may contain the entire coding sequence for the 45,000-dalton polypeptide. The 2.6-kilobase insert was subcloned into pUC8 and transformed into Escherichia coli DH5 alpha. However, the 45,000-dalton polypeptide was not detected in cell extracts of this organism when specific antibody was used. Preliminary nucleic acid sequence data correlated exactly with the amino acid sequence. A cholic acid-induced mRNA species of greater than 6 kilobases in size was identified by Northern (RNA) blot analysis of total RNA, suggesting that the gene coding for this polypeptide is part of a larger operon.     

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.     

Location of F plasmid transfer operon genes traC and traW and identification of the traW product.

As part of an analysis of the conjugative transfer genes associated with the expression of F pili by plasmid F, we have investigated the physical location of the traC and traW genes. We found that plasmid clones carrying a 2.95-kilobase EcoRI-EcoRV F transfer operon fragment were able to complement transfer of F lac traC mutants and expressed an approximately 92,000-dalton product that comigrates with TraC. We also found that traW-complementing activity was expressed from plasmids carrying a 900-base-pair SmaI-HincII fragment. The traW product was identified as an approximately 23,000-dalton protein. The two different F DNA fragments that expressed traC and traW activities do not overlap. Our data indicate that the traC gene is located in a more-tra operon promoter-proximal position than suggested on earlier maps and that traW is distal to traC. These results resolve a long-standing question concerning the relationship of traW to traC. The clones we have constructed are expected to be useful in elucidating the role of proteins TraC and TraW in F-pilus assembly.     

Evidence for a multigene family involved in bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708.     

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.     

Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

1H-15N NMR studies of Escherichia coli tRNA(Phe) from hisT mutants: a structural role for pseudouridine

Escherichia coli tRNA(Phe)U39 was isolated from a specially constructed bacterial strain (DD1003/pRK3) carrying mutations in the hisT gene (the structural gene for tRNA pseudouridine synthase I) and in the pyrB gene (uracil auxotrophy). The pheU gene for tRNA(Phe) under control of the native tRNA promoter was on a multicopy plasmid and gave up to 40-fold overproduction of tRNA(Phe)U39. The double mutant permitted efficient incorporation of [3-15N]uracil, resulting in greater than 95% 15N enrichment of uracil-derived bases. 1H and 1H-15N NMR experiments were used to assign the low-field proton resonances to specific hydrogen-bonding interactions. 1H NMR assignments indicate that tRNA(Phe)U39 has a structure similar to that of native tRNA(Phe) except in the anticodon region where replacement of pseudouridine (psi) at position 39 with uridine (U) destabilizes hydrogen-bonding interactions at the base of the anticodon stem. We propose that U----psi modifications further stabilize interactions normally available to U by providing an additional locus for hydrogen bonding to the pyrimidine ring.     

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.     

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.