A cis-acting A-U sequence element induces kinetoplastid U-insertions.
A 34-nucleotide A-U sequence located immediately upstream of the editing sites of the Leishmania tarentolae cytochrome b mRNA induces a mitochondrial extract to insert U nucleotides independent of guide RNA. Insertions are localized to positions immediately 5' and 3' of the A-U sequence. When placed within an unedited mammalian transcript, the A-U sequence is sufficient to induce U-insertions. The sequence has a high degree of similarity with the templating nucleotides of a cytochrome b guide RNA and with a sequence adjacent to the editing sites in ND7 mRNA, the other characterized kinetoplastid mRNA supporting guide RNA-independent U-insertions. At least one protein specifically interacts with the A-U sequence. The reaction is consistent with a mechanism proposed for guide RNA-directed editing. (+info
Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient.
Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mossbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear. (+info
Cloning of Bacillus stearothermophilus ctaA and heme A synthesis with the CtaA protein produced in Escherichia coli.
The Bacillus stearothermophilus ctaA gene, which is required for heme A synthesis, was found upstream of the ctaBCDEF/caaEABCD gene cluster as in B. subtilis and B. firmus. The deduced protein sequence indicate that CtaA is a 35-kDa intrinsic membrane protein with seven hydrophobic segments. Alignment of CtaA sequences showed conserved residues including histidines that may be involved in heme B binding and substrate binding. Expression of ctaA in E. coli resulted in increased formation of a membrane-bound b-type cytochrome, heme A production, and severe growth inhibition. Furthermore, B. stearothermophilus CtaA produced in E. coli was found to catalyze the conversion of heme O to heme A in vitro. (+info
Missense mutations in the gp91-phox gene encoding cytochrome b558 in patients with cytochrome b positive and negative X-linked chronic granulomatous disease.
Chronic granulomatous disease (CGD) is a disorder of host defense due to genetic defects of the superoxide (O2-) generating NADPH oxidase in phagocytes. A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the oxidase. The X-linked form of the disease is due to defects in the gp91-phox gene. We report here biochemical and genetic analyses of patients with typical and atypical X-linked CGD. Immunoblots showed that neutrophils from one patient had small amounts of p22-phox and gp91-phox and a low level of O2- forming oxidase activity, in contrast to the complete absence of both subunits in two patients with typical CGD. Using polymerase chain reactions (PCR) on cDNA and genomic DNA, we found novel missense mutations of gp91-phox in the two typical patients and a point mutation in the variant CGD, a characteristic common to two other patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient provided evidence for the presence of heme of cytochrome b558. Recently, we reported another variant CGD with similar amounts of both subunits, but without oxidase activity or the heme spectrum. A predicted mutation at amino acid 101 in gp91-phox was also confirmed in this variant CGD by PCR of the genomic DNA. These results on four patients, including those with two variant CGD, are discussed with respect to the missense mutated sites and the heme binding ligands in gp91-phox. (+info
Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase.
The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3. The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent. Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine. QPs3 was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase. Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million. The recombinant QPs3 binds ubiquinone, causing a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state. Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone. Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride. Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92. Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride. (+info
TAKs, thylakoid membrane protein kinases associated with energy transduction.
The phosphorylation of proteins within the eukaryotic photosynthetic membrane is thought to regulate a number of photosynthetic processes in land plants and algae. Both light quality and intensity influence protein kinase activity via the levels of reductants produced by the thylakoid electron transport chain. We have isolated a family of proteins called TAKs, Arabidopsis thylakoid membrane threonine kinases that phosphorylate the light harvesting complex proteins. TAK activity is enhanced by reductant and is associated with the photosynthetic reaction center II and the cytochrome b6f complex. TAKs are encoded by a gene family that has striking similarity to transforming growth factor beta receptors of metazoans. Thus thylakoid protein phosphorylation may be regulated by a cascade of reductant-controlled membrane-bound protein kinases. (+info
Ubiquinol:cytochrome c oxidoreductase. Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b.
The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N', N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0. 06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed. (+info
Atypically low rate of cytochrome b evolution in the scleractinian coral genus Acropora.
Unexpectedly low levels of mitochondrial DNA (mtDNA) cytochrome b sequence divergence are found between species of the scleractinian coral genus Acropora. Comparison of 964 positions of the cytochrome b gene of two out of the three Caribbean Acropora species with seven of their Pacific congeners shows only 0.3-0.8% sequence difference. Species in these biogeographic regions have been evolving independently for at least three million years (since the rise of the Isthmus of Panama) and this geological date is used to estimate nucleotide divergence rates. The results indicate that the Acropora cytochrome b gene is evolving at least 10-20 times slower than the 'standard' vertebrate mtDNA clock and is one of the most slowly evolving animal mitochondrial genes described to date. The possibility is discussed that, unlike higher animals, cnidarians may have a functional mtDNA mismatch repair system. (+info