Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.
The expression of the DNA modification gene (mom) of bacteriophage Mu requires the cellular deoxyadenosine methylase (dam) and a transactivation factor from the phage. By hypothesis, the transcription of mom is activated by methylation of three GATC sequences upstream from the mom gene. We have introduced small deletions at a fourth GATC site located about 140 base pairs downstream from the primary methylation region. Some of the deletions severely affect the mom gene expression. We propose from this analysis that (1) some important elements, possibly the promoter, concerned with the expression of mom are located between nucleotides 840 and 880 from the right end of Mu and (2) the mom protein starts with the codon GTG located at position 810. We favor the hypothesis that methylation turns off transcription upstream, thereby allowing the main mom promoter to function.
Transcription of the bacteriophage Mu mom operon is strongly repressed by the host OxyR protein in dam - but not dam + cells. In this work we show that the extent of mom modification is sensitive to the relative levels of the Dam and OxyR proteins and OxyR appears to modulate the level of mom expression even in dam + cells. In vitro studies demonstrated that OxyR is capable of binding hemimethylated P mom , although its affinity is reduced slightly compared with unmethylated DNA. Thus, OxyR modulation of mom expression in dam + cells can be attributed to its ability to bind hemimethylated P mom DNA, the product of DNA replication.
Arabidopsis MOM1 is required for the heritable maintenance of transcriptional gene silencing (TGS). Unlike many other silencing factors, depletion of MOM1 evokes transcription at selected loci without major changes in DNA methylation or histone modification. These loci retain unusual, bivalent chromatin properties, intermediate to both euchromatin and heterochromatin. The structure of MOM1 previously suggested an integral nuclear membrane protein with chromatin-remodeling and actin-binding activities. Unexpected results presented here challenge these presumed MOM1 activities and demonstrate that less than 13% of MOM1 sequence is necessary and sufficient for TGS maintenance. This active sequence encompasses a novel Conserved MOM1 Motif 2 (CMM2). The high conservation suggests that CMM2 has been the subject of strong evolutionary pressure. The replacement of Arabidopsis CMM2 by a poplar motif reveals its functional conservation. Interspecies comparison suggests that MOM1 proteins emerged at the origin of vascular plants through neo-functionalization of the ubiquitous eukaryotic CHD3 chromatin remodeling factors. Interestingly, despite the divergent evolution of CHD3 and MOM1, we observed functional cooperation in epigenetic control involving unrelated protein motifs and thus probably diverse mechanisms.
Epigenetic regulation of transcription usually involves changes in histone modifications, as well as DNA methylation changes in plants and mammals. Previously, we found an exceptional epigenetic regulator in Arabidopsis, MOM1, acting independently of these epigenetic marks. Interestingly, MOM1 controls loci associated with bivalent chromatin marks, intermediate to active euchromatin and silent heterochromatin. Such bivalent marks are often associated with newly inserted and/or potentially active transposons, silent transgenes, and certain chromosomal loci. Notably, bivalent chromatin seems to be characteristic for embryonic stem cells, where such loci change their activity and determination of epigenetic marks during cell differentiation. Here, we provide evidence that in vascular plants, the MOM1-like proteins evolved from the ubiquitous eukaryotic chromatin remodeling factor CHD3. The domains necessary for CHD3 function degenerated in MOM1, became dispensable for its gene silencing activity, and were replaced by a novel, unrelated domain providing silencing function. Therefore, MOM1-like proteins use a different silencing mechanism compared to the ancestral CHD3s. In spite of this divergent evolution, CHD3 and MOM1 seem to retain a functional cooperation in control of transcriptionally silent loci. Our results provide an unprecedented example of an evolutionary path for epigenetic components resulting in increased complexity of an epigenetic regulatory network characteristic for multicellular eukaryotes.
Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA–independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.
Epigenetic shifts in transcriptional activities are usually correlated with changes in chromatin properties and covalent modification of DNA and/or histones. There are, however, exceptional regulators that are able to switch epigenetic states without the apparent involvement of changes in chromatin or DNA modifications. MOM1 protein, derived from CHD3 chromatin remodelers, belongs to this group. Here we defined a very small domain of MOM1 (less than 5% of its total sequence) that is sufficient for epigenetic regulation. We solved the structure of this domain and found that it forms a dimer with each monomer consisting of unusual consecutive 11 amino-acid hendecad repeats folding into an antiparallel coiled-coil. In vivo experiments demonstrated that the formation of this coiled-coil is essential for silencing activity; however, it is effective only at loci co-silenced by MOM1 and small RNAs. At loci not controlled by small RNAs, the entire MOM1 protein is required. Our results demonstrate that a single epigenetic regulator is able to differentially use its domains to control diverse chromosomal targets. The acquisition of the coiled-coil domain of MOM1 reflects a neofunctionalization of CHD3 proteins, which allowed MOM1 to broaden its activity and to provide input into multiple epigenetic pathways.
The phage Mu C gene product is a specific activator of Mu late gene transcription, including activation of the mom operon. Fusion of the C gene to the efficient translation initiation region of the Escherichia coli atpE gene allowed significant overproduction of C protein, which was subsequently purified and assayed for DNA binding by gel retardation and nuclease footprinting techniques. C protein binds to a site immediately upstream of the -35 region both of the mom promoter and the related phage D108 mod promoter. The location of the mom promoter has been determined by primer extension. Upstream deletions extending more than 3 base pairs into the C-binding site abolished activation of the mom promoter in vivo. In vitro binding of C was not significantly affected by DNA methylation. A second, C-dependent promoter was identified just downstream of the C coding region; comparison with the mom promoter revealed common structural elements.
Background and purpose
Joint replacement with metal-on-metal (MOM) bearings have gained popularity in the last decades in young and active patients. However, the possible effects of MOM wear debris and its corrosion products are still the subject of debate. Alongside the potential disadvantages such as toxicity, the influences of metal particles and metal ions on infection risk are unclear.
We reviewed the available literature on the influence of degradation products of MOM bearings in total hip arthroplasties on infection risk.
Wear products were found to influence the risk of infection by hampering the immune system, by inhibiting or accelerating bacterial growth, and by a possible antibiotic resistance and heavy metal co-selection mechanism.
Whether or not the combined effects of MOM wear products make MOM bearings less or more prone to infection requires investigation in the near future.
Bacteriophage Mu DNA was labeled after induction in the presence of [2-3H]adenine or [8-3H]adenine. Both Mu mom+·dam+ DNA and Mu mom−·dam+ DNA have similar N6-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R·DpnI but resistant to cleavage by R·DpnII. These results indicate that the mom+ protein does not alter the sequence specificity of the host dam+ methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted Ax, in Mu mom+·dam+ DNA; approximately 15% of the adenine residues are modified to Ax. Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N6-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. Ax is not present in Mu mom+·dam+ or Mu mom−·dam+ phage DNA or in cellular DNA from uninduced Mu mom+·dam+ lysogens. These results suggest that expression of the dam+ and mom+ genes are required for the Ax modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom+·dam− DNA and Mu mom−·dam− DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5′... C-A*-C... 3′ and that this methylation is mediated by the EcoK modification enzyme.
The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.
RNA-directed modification of histones is essential for maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation, especially in non-CG sequence contexts, is tightly related to inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. We recently revealed that MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana silences endogenous loci related to transposons and homologous to the 24-nt siRNAs accumulated in wild type plants, and suggested that MOM1 transduces RNA-directed DNA methylation (RdDM) signals to repressive histone modification. In this addendum, we focus on the involvement of MOM1 in multiple transcriptional gene silencing (TGS) pathways.
Arabidopsis thaliana; RNA-directed DNA methylation; histone modification; MORPHEUS' MOLECULE 1
Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. S1 nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli sigma 70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent beta-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.
An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
The phage Mu gene C encodes a 16.5-kDa site-specific DNA-binding protein that functions as a trans-activator of the four phage "late" operons, including mom. We have overexpressed and purified C and used it for DNase I footprinting and transcription analyses in vitro. The footprinting results are summarized as follows. (i) As shown previously (V. Balke, V. Nagaraja, T. Gindlesperger, and S. Hattman, Nucleic Acids Res. 12:2777-2784, 1992) in vivo, Escherichia coli RNA polymerase (RNAP) bound the wild-type (wt) mom promoter at a site slightly upstream from the functionally active site bound on the C-independent tin7 mutant promoter. (ii) In the presence of C, however, RNAP bound the wt promoter at the same site as tin7. (iii) C and RNAP were both bound by the mom promoter at overlapping sites, indicating that they were probably on different faces of the DNA helix. The minicircle system of Choy and Adhya (H. E. Choy and S. Adhya, Proc. Natl. Acad. Sci. USA 90:472-476, 1993) was used to compare transcription in vitro from the wt and tin7 promoters. This analysis showed the following. (i) Few full-length transcripts were observed from the wt promoter in the absence of C, but addition of increasing amounts of C greatly stimulated transcription. (ii) RNA was transcribed from the tin7 promoter in the absence of C, but addition of C had a small stimulatory effect. (iii) Transcription from linearized minicircles or restriction fragment templates was greatly reduced (although still stimulated by C) with both the wt and tin7 promoters. These results show that C alone is capable of activating rightward transcription in vitro by promoting RNAP binding at a functionally active site. Additionally, DNA topology plays an important role in transcriptional activation in vitro.
The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors.
Establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the PRM promoter and repress transcription from the divergent PR promoter. Several lines of evidence indicate that the 434 repressor activates initiation of PRM transcription by occupying a binding site adjacent to the PRM promoter and directly contacting RNA polymerase. The overlapping architecture of the PRM and PR promoters suggests that an RNA polymerase bound at PR may repress PRM transcription initiation. Hence, part of the stimulatory effect of the 434 repressor may be relief of interference between RNA polymerase binding to the PRM promoter and to the PR promoter. Consistent with this proposal, we show that the repressor cannot activate PRM transcription if RNA polymerase binds at PR prior to addition of the 434 repressor. However, unlike the findings with the related λ phage, formation of RNA polymerase promoter complexes at PRM and at PR apparently are mutually exclusive. We find that the RNA polymerase-mediated inhibition of repressor-stimulated PRM transcription requires the presence of an open complex at PR. Taken together, these results indicate that establishment of an open complex at PR directly prevents formation of an RNA polymerase-PRM complex.
Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24B prophage is proposed.
The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.
Temperate bacteriophage parasitize their host by integrating into the host genome where they provide additional genetic information that confers higher fitness on the host bacterium by protecting it against invasion by other bacteriophage, by increasing serum resistance, and by coding for toxins and adhesion factors that help the parasitized bacterium invade or evade its host. Here we ask if a temperate phage can also regulate host genes. We find several different host functions that are down-regulated in lysogens. The pckA gene, required for gluconeogenesis in all living systems, is regulated directly by the principal repressor of many different temperate prophage, the cI protein. cI binds to the regulatory region of pckA, thereby shutting down pckA transcription. The pckA regulatory region has target sequences for many other temperate phage repressors, and thus we suggest that down-regulation of the host pckA pathway increases lysogen fitness by lowering the growth rate of lysogens in energy-poor environments, perhaps as an adaptive response to the host predation system or as an aspect of lysogeny that must be offset by down-regulating pckA.
Lysogenic bacteriophage such as lambda integrate into their host genome, but do they regulate specific host genes? This study shows that they do, thereby increasing the fitness of the lysogen.
Bacteriophage classification is mainly based on morphological traits and genome characteristics combined with host information and in some cases on phage growth lifestyle. A lack of molecular tools can impede more precise studies on phylogenetic relationships or even a taxonomic classification. The use of methods to analyze genome sequences without the requirement for homology has allowed advances in classification.
Here, we proposed to use genome sequence signature to characterize bacteriophages and to compare them to their host genome signature in order to obtain host-phage relationships and information on their lifestyle. We analyze the host-phage relationships in the four most representative groups of Caudoviridae, the dsDNA group of phages. We demonstrate that the use of phage genomic signature and its comparison with that of the host allows a grouping of phages and is also able to predict the host-phage relationships (lytic vs. temperate).
We can thus condense, in relatively simple figures, this phage information dispersed over many publications.
The PARKIN (PARK2) ubiquitin ligase and its regulatory kinase PINK1 (PARK6), often mutated in familial early onset Parkinson’s Disease (PD), play central roles in mitochondrial homeostasis and mitophagy.1–3 While PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM,1,4–11 the full repertoire of PARKIN substrates – the PARKIN-dependent ubiquitylome - remains poorly defined. Here we employ quantitative diGLY capture proteomics12,13 to elucidate the ubiquitylation site-specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN’s active site residue C431, which has been found mutated in PD patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.
The mitochondrial outer membrane contains import receptors for preproteins and a multisubunit general insertion pore. Several small outer membrane proteins (< 10 kDa) have been identified by their association with receptors or the general insertion pore, yet little is known about their function. Here, we present evidence that the biochemically identified Mom8b and the genetically identified Isp6 are identical. A deletion of Mom8b/Isp6 in Saccharomyces cerevisiae leads to (i) a delay of import of preproteins, (ii) stabilization of preprotein binding to receptors and the general insertion pore, and (iii) destabilization of the interaction between receptors and the general insertion pore. These results suggest that Mom8b supports the cooperativity between receptors and the general insertion pore and facilitates the release of preproteins from import components and thereby promotes efficient transfer of preproteins.
We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.
Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coli RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase I-sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i) in vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from -74 to -24 with respect to transcription initiation. This overlaps a known strong C-binding site (at -35 to -54). RNAP bound at P2 appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to +21. With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from -35 to -54), RNAP bound to P1 independent of C. We conclude that P1 is the 'functional' RNAP binding site for mom-transcription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.
Late in its growth cycle, transcription of the phage Mu mom promoter (Pmom) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the α or ς70 subunit of Escherichia coli RNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both α and ς70.
Mitochondrial outer membrane (MOM) proteins in parasitic protozoa like Trypanosoma brucei are poorly characterized. In fungi and higher eukaryotes, Tob55 is responsible for the assembly of β-barrel proteins in the MOM. Here we show that T. brucei Tob55 (TbTob55) has considerable similarity in its primary and secondary structure to Tob55 from other species. TbTob55 is localized in T. brucei MOM and is essential for procyclic cell survival. Induction of Tob55 RNAi decreased the level of the voltage-dependent anion channel (VDAC) within 48 h. Although the primary effect is on VDAC, induction of TbTob55 RNAi for a longer time period also decreased the levels of other nucleus encoded mitochondrial proteins. In addition, the mitochondrial membrane potential was reduced at this later time point possibly due to a reduction in the level of the proteins involved in oxidative phosphorylation. However, mitochondrial structure was not altered due to depletion of Tob55. In vitro protein import of VDAC into mitochondria with a 50-60% reduction of TbTob55 was reduced about 40% in comparison to uninduced control. In addition, the import of presequence-containing proteins such as, cytochrome oxidase subunit 4 (COIV) and trypanosome alternative oxidase (TAO) was affected by about 20 % under this condition. Depletion of VDAC levels by RNAi did not affect the import of either COIV or TAO. Furthermore, TbTob55 over expression increased the steady state level of VDAC as well as the level of the assembled protein complex of VDAC, suggesting that similar to other eukaryotes TbTob55 is involved in assembly of MOM β-barrel proteins and plays an indirect role in the biogenesis of mitochondrial preproteins destined for the mitochondrial inner membrane.
Tob55; Trypanosoma brucei; RNAi; VDAC biogenesis; mitochondrial protein import