The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence.

The yeast and mammalian branchpoint sequence binding proteins (BBP and mBBP/SF1) contain both KH domain and Zn knuckle RNA-binding motifs. The single KH domain of these proteins is sufficient for specific recognition of the pre-mRNA branchpoint sequence (BPS). However, an interaction is only apparent if one or more accessory modules are present to increase binding affinity. The Zn knuckles of BBP/mBBP can be replaced by an RNA-binding peptide derived from the HIV-1 nucleocapsid protein or by an arginine-serine (RS)7 peptide, without loss of specificity. Only the seven-nucleotide branchpoint sequence and two nucleotides to either side are necessary for RNA binding to the chimeric proteins. Therefore, we propose that all three of these accessory RNA-binding modules bind the phosphate backbone, whereas the KH domain interacts specifically with the bases of the BPS. Proteins and protein complexes with multiple RNA-binding motifs are frequent, suggesting that an intimate collaboration between two or more motifs will be a general theme in RNA-protein interactions.     

The physiologic role of pyocyanine synthesized by Pseudomonas aeruginosa

The physiological role of pyocyanine for Pseudomonas aeruginosa was studied. Its synthesis was shown to commence at the retardation growth phase. Pyocyanine was accumulated only in the growth medium. The addition of 2,6-dichlorophenolindophenol accepting the reducing equivalents from coenzyme Q and transferring them to cytochrome c inhibited the pigment accumulation. This was indicative of the connection between pyocyanine synthesis and the level of the reducing equivalents in the cells. Pyocyanine did not accept the reducing equivalents from coenzyme Q in the respiratory chain of P. aeruginosa. Only reduced pyridine nucleotides served as substrates for pyocyanine in the reaction of autooxidation. The kinetic parameters of this reaction and the affinity of NADH dehydrogenase for the substrate were measured. The kinetic data were analysed to show that, under the physiological conditions, pyocyanine could not apparently compete with the respiratory chain for the reducing equivalents and hence directly regulate the level of NAD(P)H in P. aeruginosa cells. In order to keep the oxidising activity at a level necessary for the cells, the latter decreased the content of the reducing equivalents either by synthesizing pyocyanine or owing to the activity of cyanide-resistant oxidase. These processes of releasing the reducing equivalents are in a reciprocal relationship.     

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large‐scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe²⁺ or Mg²⁺ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen‐regulated pufQBALMX operon encoding the reaction centre–light‐harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG‐immunoprecipitation experiments retrieve a ferrochelatase‐PufQ‐carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen‐limiting conditions. The co‐location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light‐harvesting polypeptides.     

Mechanism of the antibiotic action pyocyanine.

Exposure of Escherichia coli growing in a rich medium to pyocyanine resulted in increased intracellular levels of superoxide dismutase and of catalase. When these adaptive enzyme syntheses were prevented by nutritional paucity, the toxic action of pyocyanine was augmented. The antibiotic action of pyocyanine was dependent upon oxygen and was diminished by superoxide dismutase and by catalase, added to the suspending medium. Pyocyanine slightly augmented the respiration of E. coli suspended in a rich medium, but greatly increased the cyanide-resistant respiration. Pyocyanine was able to cause the oxidation of reduced nicotinamide adenine dinucleotide, with O2- production, in the absence of enzymatic catalysis. It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.     

Possibility of detecting pyocyanine in Pseudomonas aeruginosa cells

Pyocyanin can be detected in the cells of Pseudomonas aeruginosa using UV and IR spectroscopy of disturbed complete inner reflection (DCIR). Intact cells of the parent strain liberating the pigment into the cultural broth and mutant cells lacking the ability contain pyocyanin within the cells. Occasionally, pyocyanin can be detected in the outer layers of the cells, which is more typical of the parent strain. In the freshly isolated fractions of the parent strain cellular walls, pyocyanin seems to be pesent in the bound state that has changed significantly its structural organization. In due course, the hypothetical complex pyocyanin--cellular wall decomposes to yield an "oxidized" pigment similar to that liberated into the cultural broth. the cell wall of the mutant possesses the properties of "oxidized" pyocyanin immediately after isolation of the fraction. The pigment cannot be identified in the fractions of cytoplasmic membranes; pyocyanin is present in the "oxidized" state in the fractions of cytoplasm for the cells of both types. The paper discusses the role of the permeability of cytoplasmic membranes in the transport of pyocyanin from the cytoplasm into the cellular wall of the bacterium and then into the surrounding medium.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Phenotype recognition of pyocyanine mutants in pseudomonas aeruginosa

Certain classes of pyocyanine mutants in Pseudomonas aeruginosa escape detection when screened in the presence of wild-type or other mutant cells. A technique is described for recognizing mutant phenotypes after cells are in individual agar wells. The procedure eliminates cross-feeding phenomena as well as the masking of mutant phenotypes by diffusing pyocyanine produced by nearby clones.     

Alterations of inosinate branchpoint enzymes in cultured human lymphoblasts

The specific activities of the three enzymes of the inosinate branchpoint are independently regulated when lymphoblasts are grown under various tissue culture conditions. In comparison to rapidly dividing cells, lymphoblasts at high cell density with no cellular division have decreased activity of the enzymes which commit inosinate to adenylate or guanylate, while cytoplasmic 5'-nucleotidase is relatively preserved. A linear relationship between inosinate dehydrogenase activity and growth rate (r = 0.92) exists in lymphoblasts with slowed growth rates. In contrast, in dividing cells adenylosuccinate synthetase and 5'-nucleotidase do not vary with growth rate. Adenylosuccinate synthetase and inosinate dehydrogenase activities appear to be related to the presence or rate of cellular division, as opposed to the presence or degree of neoplastic transformation. Lymphoblast lines with alterations of specific purine metabolic enzymes have characteristic alteration of the inosinate utilizing enzymes. Deficiencies of purine nucleoside phosphorylase or hypoxanthine phosphoribosyltransferase, abnormalities which render the cell unable to salvage purine effectively, are associated with depressed inosinate dehydrogenase activity. Insertion of the hypoxanthine phosphoribosyltransferase gene into hypoxanthine phosphoribosyltransferase-deficient cells normalizes inosinate dehydrogenase activity, while a hypoxanthine phosphoribosyltransferase-deficient mutant selected from a hypoxanthine phosphoribosyltransferase-containing line has depressed inosinate dehydrogenase activity. In contrast, overactivity of phosphoribosylpyrophosphate synthetase, with enhanced excretion of purines due to excessive production, is associated with elevated inosinate dehydrogenase activity. Inosinate dehydrogenase appears to be regulated according to the availability of purine nucleotides. Patients who overproduce uric acid and potentially have undescribed purine metabolic defects are now being screened for abnormalities in the inosinate branchpoint enzymes.