In vivo incorporation of an alkyne into proteins in Escherichia coli

Using a genetic selection we identified mutants of the M. janaschii tyrosyl-tRNA synthetase that selectively charge an amber suppressor tRNA with para-propargyloxyphenylalanine in Escherichia coli. These evolved tRNA-synthetase pairs were used to site-specifically incorporate an alkynyl group into a protein, which was subsequently conjugated with fluorescent dyes by a [3+2]-cycloaddition reaction under mild reaction conditions.     

Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle). Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10-12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3). Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.     

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.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.     

In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells

19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells. 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo. By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C.     

Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid.

A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wild-type cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.     

Photochemistry of 4-thiouridine in Escherichia coli transfer RNA1Val

Irradiation of pure transfer RNA₁^Val with monochromatic light (334 nm) produces characteristic changes in the spectral properties of 4-thiouridine, the only base which strongly absorbs light at this wavelength. Variations in absorption and fluorescence of 4-thiouridine during irradiation are interpreted in terms of a specific, quantitative photoreaction which proceeds with a yield of about 5 × 10⁻³E/mole. The photoreaction occurs under conditions where tRNA₁^Val is biologically active but not under conditions that destroy the tertiary structure of the 4-thiouridine region.     

Zinc uptake and incorporation into proteins in T4D bacteriophage-infected Escherichia coli.

The metabolism of Zn2+ in Escherichia coli infected with T4D bacteriophage and various T4D mutants has been examined. E. coli B infected with T4D, and all T4D mutants except T4D 12-, took up zinc ions at a rate identical to that of uninfected cells. E. coli B infected with T4D 12- had a markedly decreased rate of zinc uptake. The incorporation of zinc into proteins of infected cells has also been studied. T4D phage infection was found to shut off the synthesis of all bacterial host zinc metalloproteins while allowing the formation of viral-induced zinc proteins. The amount of zinc incorporated into viral proteins was affected by the absence of various T4D gene products. Cells infected with T4D 12-, and to a much less extent those infected with T4D 29-, incorporated the least amount of zinc into proteins, while cells infected with T4D 11- and T4D 51- incorporated increased amounts of zinc into the zinc metalloproteins. In cells infected with T4D 11- and 51- most of the zinc protein was found to be the product of gene 12. The marked effect of infection of E. coli with T4D 12- on both zinc uptake and zinc incorporation into protein supports the conclusion that T4D gene 12 protein is a zinc metalloprotein. Additionally, these observations have indicated that this metalloprotein interacts with host cell membrane.     

In vivo regulation of the Escherichia coli araC promoter.

The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose. This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions. In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible. Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants. Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.     

In vivo incorporation of cytidine-monophosphate-ciliatine into rat liver lipids.

1. In vivo this investigation was carried out in order to compare the incorporation into rat lipids of free [1,2-minus 14C]-ciliatine and CMP-[1,2-minus 14C]-ciliatine which is the precursor in phosphonolipid biosynthesis. 2. The incorporation of the radioactivity from CMP-[1,2-minus 14C]-ciliatine took place more rapidly than that from free [1,2-minus 14C]-ciliatine in both liver and kidney. The amount of radioactivity from the CMP-[1,2-minus 14C]-ciliatine incorporated into total liver lipids was about 5 times higher than that incorporated into total liver lipids of rat two hrs after injecting free-[1,2-minus 14C]-ciliatine. 3. The amount of [1,2-minus 14C]-ciliatine incorporated into total liver lipids was 15 and 21 times higher than that incorporated into total kidney lipids of rat two and four hrs after injecting free [1,2-minus 14C]-ciliatine. 4. If the main pathway for the phosphonolipid biosynthesis is via CMP-ciliatine, the rate of phosphonolipid formation from CMP-ciliatine must therefore be higher than that from free-ciliatine. The results obtained here indicate therefore that the main pathway for phosphonolipid biosynthesis is a pathway involving CMP-ciliatine. 5. An unknow compound was detected in the water soluble fraction of the acid hydrolyzate of liver phosphonolipids. This material migrated with the N-trimethyl-derivative of ciliatine on the thin-layer chromatogram. The result shows that there is therefore a possibility of methylation of exogenous ciliatine to the phosphonate analogue of choline in the mammalian body.     

Specific incorporation of 5-fluorocytidine into Escherichia coli RNA

RNAs isolated from Escherichia coli B grown in the presence of 5-fluorouracil have high levels of the analog replacing uridine and uridine-derived modified nucleosides. Cytidine has also been shown to be replaced in these RNAs by 5-fluorocytidine, a metabolic product of 5-fluorouracil, but to a considerably lesser extent. When 5-fluorocytidine is added to cultured of E. coli B little 5-fluorocytidine (0.20 mol%) is incorporated into cellular RNAs because of the active cytosine/cytidine deaminase activities. Addition of the cytidine deaminase inhibitor tetrahydrouridine (70 micrograms/ml) increases 5-fluorocytidine incorporation to about 3 mol% in tRNAs, but does not eliminate 5-fluorouridine incorporation. E. coli mutants lacking cytosine/cytidine deaminase activities are able to more than double the extent of 5-fluorocytidine incorporation into their transfer and ribosomal RNAs, replacing cytidine with no detectable 5-fluorouridine incorporation. Levels of 5-methyluridine, pseudouridine and dihydrouridine in tRNAs are not affected. These fluorocytidine-containing tRNAs show amino acid-accepting activities similar to control tRNAs. Fluorocytidine was found to be quite susceptible to deamination under alkaline conditions. Its conversion to primarily 5-fluorouridine follows pseudo-first-order reaction kinetics with a half-life of 10 h in 0.3 M KOH at 37 degrees C. This instability in alkali probably explains why 5-fluorocytidine was not found earlier in RNAs isolated from cells treated with 5-fluorouridine, since most early RNA hydrolyses were carried out in alkali. It may also explain the mild mutagenic properties observed in some systems following 5-fluorouridine treatment. Initial 19F-NMR measurements in fluorocytidine-containing tRNAs indicate that this modified tRNA may be useful in future structural studies of tRNAs and in probing tRNA-protein complexes.     

In vivo regulation of chromosomal beta-lactamase in Escherichia coli.

Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo. Both the activity and the amount of beta-lactamase increased with growth rate. During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation. After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min. To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature. No influence by the relA gene product was found on beta-lactamase synthesis. The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.     

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.