DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region.

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction.

Transposase-DNA Complex Structures Reveal Mechanisms for Conjugative Transposition of Antibiotic Resistance.

Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.

Conjugative transposition of the vancomycin resistance carrying Tn1549: enzymatic requirements and target site preferences.

Rapid spread of resistance to vancomycin has generated difficult to treat bacterial pathogens worldwide. Though vancomycin resistance is often conferred by the conjugative transposon Tn1549, it is yet unclear whether Tn1549 moves actively between bacteria. Here we demonstrate, through development of an in vivo assay system, that a mini-Tn1549 can transpose in E. coli away from its natural Gram-positive host. We find the transposon-encoded INT enzyme and its catalytic tyrosine Y380 to be essential for transposition. A second Tn1549 protein, XIS is important for efficient and accurate transposition. We further show that DNA flanking the left transposon end is critical for excision, with changes to nucleotides 7 and 9 impairing movement. These mutations could be partially compensated for by changing the final nucleotide of the right transposon end, implying concerted excision of the two ends. With changes in these essential DNA sequences, or without XIS, a large amount of flanking DNA transposes with Tn1549. This rescues mobility and allows the transposon to capture and transfer flanking genomic DNA. We further identify the transposon integration target sites as TTTT-N6-AAAA. Overall, our results provide molecular insights into conjugative transposition and the adaptability of Tn1549 for efficient antibiotic resistance transfer.

Protein Flexibility and Synergy of HMG Domains Underlie U-Turn Bending of DNA by TFAM in Solution.

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA.

The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein.

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria.

U-turn DNA bending by human mitochondrial transcription factor A.

Transcription factor A (TFAM) is involved in the transcription regulation, maintenance and compaction of the mitochondrial genome. Recent structural data on TFAM showed its mode of operation and clarified previous biochemical and genetic results. In solution, TFAM is highly dynamic. According to crystal structures of its complex with the cognate light-strand promoter (LSP) binding sequence, it intertwines and dramatically bends DNA, thereby allowing interactions with the transcription initiation machinery. Recent studies have shown TFAM sliding on non-specific DNA, which induces compaction by increasing DNA flexibility. Finally, the structural localization of disease-related TFAM mutations suggests functional impairment at the molecular level.

Human mitochondrial transcription factor A induces a U-turn structure in the light strand promoter.

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.

Human mitochondrial mTERF wraps around DNA through a left-handed superhelical tandem repeat.

The regulation of mitochondrial DNA (mtDNA) processes is slowly being characterized at a structural level. We present here crystal structures of human mitochondrial regulator mTERF, a transcription termination factor also implicated in replication pausing, in complex with double-stranded DNA oligonucleotides containing the tRNA(Leu)(UUR) gene sequence. mTERF comprises nine left-handed helical tandem repeats that form a left-handed superhelix, the Zurdo domain.