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.     

Specific incorporation of selenium into lysine- and glutamate- accepting tRNAs from Escherichia coli

Amino acid transfer nucleic acids (tRNAs) that contain selenium-modified bases are synthesized by Escherichia coli in the presence of low levels (0.1-0.5 microM) of [75Se]selenite or [75Se]selenate. The amount of selenium incorporated (1-2 g atoms/100 mol of tRNA) was unchanged by 10-20-fold variations in selenium or sulfate concentrations or by the addition of 1 mM cysteine, sulfide, or sulfite. Specific incorporation of selenium (as opposed to nonspecific substitution for sulfur) was further indicated by the different reversed phase chromatographic elution patterns of 35S- and 75Se-labeled tRNAs isolated from cells labeled with 35SO2-4 and 75SeO2-4. Also, E. coli mutants unable to synthesize an abundant sulfur-modified base, 4-thiouracil, nevertheless produced normal levels of selenium-modified tRNAs. Two different methods of distinguishing between aminoacylated and nonaminoacylated tRNA, one which examined mobility during reversed phase chromatography and another which employed anti-AMP antibodies, indicated that over 50% of the selenium-containing tRNA had lysine or glutamate acceptor activity.     

Deuterium incorporation into Escherichia coli proteins. A neutron-scattering study of DNA-dependent RNA polymerase

Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements.     

Biosynthesis and incorporation into protein of norleucine by Escherichia coli

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.     

Heterogeneity of 5S RNA in Escherichia coli

Summary From finger-print analysis of 5S RNA isolated from 4 different Escherichia coli strains, heterogeneity of this RNA species is shown. Results point to redundancy for 5S RNA cistrons.     

Fluorescence modification of Escherichia coli 5S RNA

Reaction of 5S RNA with chlorocetaldehyde leads to the conversion of unpaired adenines to the fluorescent 1,N6-etheno-adenine derivatives. Up to 16 of the 23 adenines in free 5S RNA can be modified, the fastest reacting are A29, A34, A57-59. Partial modification of adenines in this area results in a 20% reduction in the efficiency of 5S RNA incorporation into 50S subunits during reconstitution and a 15% reduction in the activity of these subunits in peptide synthesis. Fluorescence from 1,N6-etheno-adenine is quenched in free 5S RNA and is not detectably further influenced by the binding of proteins E-L5, E-L18 and E-L25, nor by the first stage of the two step E. coli 50S subunit reconstitution procedure. However, the fluorescence is further reduced to near zero after the second step of the reconstitution. Thus, 5S RNS free in solution contains 16 unpaired adenines, those in the region between A29 and A59 particularly accessible to modification by chlorocetaldehyde. This portion of the 5S RNA molecule appears to undergo either a conformational change or interacts with other ribosomal components in the last stage of subunit reassembly.     

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.     

Efficient incorporation of unnatural amino acids into proteins in Escherichia coli

We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.     

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.     

Specific polyadenylation and purification of total messenger RNA from Escherichia coli.

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.     

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.     

Sensitization of Escherichia coli C to gamma-radiation by 5-bromouracil incorporation.

Escherichia coli C cells, unifilarly substituted with 5-bromouracil (BrUra) were 2-25 times as sensitive as unsubstituted cells to killing by gamma-irradiation under aerobic conditions. The yield of DNA double-strand breaks in BrUra-substituted cells was increased by a factor only 1-55, suggesting that other lesions also contribute to cell-killing. Alkaline sucrose density gradient analysis of the 3H-thymine labelled DNA strand showed there was less repair of gamma-ray-induced single-strand breaks when BrUra was in the complementary strand. Since there are more of these unrepaired breaks than can be accounted for by BrUra-induced DNA double-strand breakage, some fraction of the lethal events in BrUra-substituted E. coli cells may be unrepaired DNA single-strand breaks.