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Mitochondria are vital organelles regulating essential cellular functions. Human mitochondrial DNA (mtDNA) consists of 37 genes, 13 of which encode mitochondrial proteins, and the remaining 24 genes encode two ribosomal RNAs and 22 transfer RNAs needed for the translation of the mtDNA‐encoded 13 proteins. However, mtDNA often impairs the expression and function of these genes due to various mutations, ultimately causing mitochondrial dysfunction. To recover from this desperate condition, developing the technology to supply all mitochondrial proteins encoded by mtDNA at once is an urgent task, but there is no established strategy for this purpose. In this study, a simple yet effective mitochondrial gene delivery system is proposed comprising an artificial peptide inspired by a transmembrane mitochondrial membrane protein. The designed mitochondria‐targeting peptides presented on the carrier surface effectively guide the encapsulated plasmid to the mitochondria, facilitating mitochondrial uptake and gene expression. The developed system successfully delivers exogenous mtDNA to mtDNA‐depleted cells and leads to simultaneous multigene expression, ultimately restoring mitochondrial functions, including the mitochondrial respiration rate. The established multiple gene expression system in each mitochondrion is a game‐changing technology that can accelerate the development of mitochondrial engineering technologies as well as clinical applications for mitochondrial diseases. [ABSTRACT FROM AUTHOR]

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter- AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP- DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA- N44 A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA- ATP hydrolysis during RIDA. [ABSTRACT FROM AUTHOR]

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp. [ABSTRACT FROM AUTHOR]

The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE ( trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio-6-iodeadenosine, indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification. [ABSTRACT FROM AUTHOR]

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. [ABSTRACT FROM AUTHOR]

Focuses on the DNA replication-coupled inactivation of Dna protein in vitro. Initiation of chromosomal replication in Escherichia coli; Outcome of enhanced clamp formation; Conservation of arginine residue among Dna homologues.