ToxR (RegA) activates Escherichia coli RNA polymerase to initiate transcription of Pseudomonas aeruginosa toxA.

The Pseudomonas aeruginosa (Pa) structural gene (toxA), which encodes the exotoxin A protein has been shown to be regulated at the transcriptional level by a protein designated ToxR (also known as RegA). We have previously reported that ToxR directly enhances toxA transcription in vitro; however, in the absence of ToxR, Pa RNA polymerase (RNAP) transcribes toxA with low efficiency. In the present study, we have examined the ability of ToxR to initiate toxA transcription using the heterologous Escherichia coli (Ec) RNAP and found that ToxR can function with Ec RNAP to efficiently transcribe toxA both in vitro and in vivo. Antibodies produced against the sigma 70 subunit of Ec RNAP inhibit ToxR-mediated enhancement of toxA transcription, suggesting that the RNAP holoenzyme (E sigma 70) is required for transcriptional activation of toxA. We further demonstrate that ToxR is required for open-complex formation at the toxA promoter. By selectively deleting toxA upstream sequences, we have localized at 214-bp region containing both the toxA promoter and a putative ToxR-binding site sufficient for ToxR-mediated transcription of toxA.

ToxR (RegA)-mediated in vitro transcription of Pseudomonas aeruginosa toxA.

Exotoxin A (ETA) has been described as a major virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa. The transcription of the ETA structural gene (toxA) has been shown to be positively regulated by the product of the toxR gene (also called regA). However, the mechanism by which ToxR regulates toxA transcription is still under investigation. We have expressed toxR in Escherichia coli under the control of the T7 promoter and purified the wild-type ToxR protein. We have also produced ToxR as a fusion protein consisting of the first 12 amino acids of the T7 capsid protein attached to the N terminus of the intact ToxR protein. In the present study we have developed and used an in vitro transcription assay in order to investigate the mechanism of ToxR-mediated transcriptional regulation of toxA. Under the conditions of this in vitro assay toxA transcription requires the toxR product in addition to P. aeruginosa RNA polymerase (RNAP). Both the native and the T7::ToxR fusion proteins facilitate initiation of toxA transcription in vitro in the presence of Pseudomonas RNAP. Additional studies using (i) specific enzyme-linked immunosorbent assay; (ii) indirect immunoprecipitation; and (iii) gel-filtration chromatography, indicate that ToxR binds to the purified Pseudomonas RNAP and strengthens the possibility that ToxR may be an alternative sigma factor. Furthermore, the ToxR-mediated transcription of toxA is increased approx. threefold in the presence of crude cytoplasmic extracts from P. aeruginosa ToxR+ or ToxR-RegB- strains, indicating that additional factors play a role in the efficient and optimal transcription of toxA.

Mechanism of action of developmentally regulated sea urchin inhibitor of eIF-4.

The developmentally regulated inhibitor of eIF-4 function found in unfertilized sea urchin eggs has been partially purified and its mechanism of action studied in vitro using purified recombinant eIF-4 alpha and cell-free translation systems. The results demonstrate that although the phosphorylation of eIF-4 alpha is necessary to promote protein synthesis, it is not sufficient to maintain all aspects of eIF-4 function. The egg inhibitor does not change eIF-4 alpha phosphorylation state. During the blockage of initiation caused by the egg inhibitor, eIF-4 alpha remains phosphorylated but accumulates in a 48S initiation intermediate. This suggests that the egg inhibitor functions by preventing the release of eIF-4 alpha from the small ribosomal subunit. The characteristics of the inhibitor in a reticulocyte translation system demonstrate that eIF-4 activity is inhibited within 3-6 min. However, the inhibitor's characteristics in a mRNA-dependent translation system contrast with this. Preincubation with the inhibitor for 5-25 min prior to the addition of mRNA does not prevent endogenous eIF-4 from participating in translation but diminishes its ability to be reutilized, consistent with the accumulation of eIF-4 alpha on the small ribosomal subunit. The ribosomal localization of the inhibitor suggests that it could prevent eIF-4 alpha release by direct binding. The gradual inactivation of the inhibitor following fertilization indicates that it represents a component of a novel regulatory cascade that modulates eIF-4 activity.

Estrogen induction of alcohol dehydrogenase in the uropygial gland of mallard ducks.

Treatment of mallard ducks with estradiol, or a combination of estradiol and thyroxine, has been shown to result in the proliferation of peroxisomes and production of diesters of 3-hydroxy fatty acids, the female pheromones, in the uropygial gland of male and female mallard ducks. Such a treatment results in the induction of a unique set of proteins. A cDNA library enriched in hormone-induced transcripts was subjected to differential screening. The nucleotide sequence of one of the two unique cDNA clones, DGH1, had high similarity to the Human class I alcohol dehydrogenase (ADH) gamma subunit and represented the carboxy-terminus of the protein from amino acid 190-374. SDS/PAGE and Western blot analysis of the proteins indicated that the level of a 38-kDa protein that cross-reacted with antibodies prepared against the chicken ADH was increased 5-7-fold by hormone treatment. Assays for ADH activity in the uropygial gland extracts of male mallards showed a 5-7-fold induction of the enzyme by hormone treatment. The 1.9-kb ADH mRNA levels were increased 12-14-fold under these conditions. Of all the tissues tested, the uropygial gland had the highest levels of ADH mRNA. Induction of ADH by estradiol treatment occurred only in this tissue. Elevated levels of ADH were also observed in the glands of male mallards in eclipse, the post-nuptial condition when the hormonal balance is shifted to higher estrogen levels, suggesting that this enzyme is regulated by estrogens in this period. Estradiol treatment caused an 80% decrease in the NAD+/NADH ratio in the uropygial gland and a twofold increase in the fatty alcohol oxidation rate catalyzed by the gland extract. These observations could help explain how increased levels of ADH could contribute to the production of the diesters.

In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E.

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.

Immunological detection of the messenger RNA cap-binding protein.

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.

S-acylated residues of the acyl-carrier protein subunit of Klebsiella aerogenes citrate lyase.

Oxidation of the isolated deacetyl acyl-carrier protein subunit of citrate lyase from Klebsiella aerogenes with Cu2+-o-phenanthroline complex leads exclusively to intrapeptide disulfide bridge formation indicating that the cysteamine and the cysteine residues are located in close proximity. The S-acetylation of the cysteine residue in deacetyl acyl-carrier protein subunit is catalysed by a citrate lyase ligase preparation in presence of acetate and ATP. Reaction-inactivation of citrate lyase results in deacetylation of the S-acetyl cysteamine residue of the prosthetic group but not of the S-acylated cysteine residue in the acyl-carrier protein.

Regenerating liver in aged rats produces unaltered phosphoglycerate kinase.

Pure phosphoglycerate kinase from old rat liver shows altered properties. However, when old animals are partially hepatectomized, the regenerating liver produces the "young" form of the enzyme. By 4 or 5 days after hepatectomy, the enzyme once again becomes "old" in its properties. The results are interpreted as showing that normal enzyme is produced that becomes post-synthetically modified. This interpretation agrees with the idea that in old animals, "young" enzymes become conformationally modified, perhaps because of a slowing of protein turnover.

The effect of aging on rat liver phosphoglycerate kinase and comparison with the muscle enzyme.

Pure liver phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) from old rats has been found to be an altered enzyme with certain properties which are dissimilar to those of the enzyme obtained from young animals. Stability during storage, sensitivity to heat, response to antiserum and stability during isoelectric focusing differ. Unchanged are molecular weight, specific activity, Km, reactivity of SH groups, blocked N-terminal residue and leucine for the C-terminal residue. Liver phosphoglycerate kinase differs substantially from the muscle enzyme. Among the differences are stability, heat-sensitivity, Km for 3-phosphoglyceric acid, inactivation by urea and response to antiserum. Nonetheless, a number of properties suggest that the liver and muscle enzymes are similar in structure. Both react with antisera prepared to phosphoglycerate kinase from muscle and liver, respectively. For both enzymes, the N-terminal residue is blocked and the C-terminal amino acid is leucine. The muscle form has been named phosphoglycerate kinase-1 or A. The only previously known isozyme is the testis enzyme, phosphoglycerate kinase-2 or B. We therefore propose that the liver enzyme be known as phosphoglycerate kinase-3 or C.

The factor which aggregates nematode, yeast and liver phosphoglycerate kinase in tRNA.

1. A factor isolated from the free-living nematode, Turbatrix aceti and from yeast, causes aggregation of phosphoglycerate kinase from nematodes, yeast and rat liver. The rat muscle enzyme is not affected. 2. The aggregation factor is either identical to or very similar to tRNA, Pure tRNA from yeast or Escherichia coli, when mixed with nematode, yeast or rat liver phosphoglycerate kinase causes the enzyme to aggregate to higher mol. wt forms. Both the natural factor and tRNA bring about similar changes in the behavior of nematode phosphoglycerate kinase. 3. The tRNA does not remain bound to the enzyme though it appears to cause a sequential aggregation from monomer to tetramer. 4. No conclusion could be reached as to whether the factor plays a physiological role or if it is simply tRNA fortuitously present during purification of the enzyme.