Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent-LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains-LT2 and 14028S-for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems.

The portal protein of tailed bacteriophage plays essential roles in various aspects of capsid assembly, motor assembly, genome packaging, connector formation, and infection processes. After DNA packaging is complete, additional proteins are assembled onto the portal to form the connector complex, which is crucial as it bridges the mature head and tail. In this study, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the portal vertex from bacteriophage lambda in both its prohead and mature virion states. Comparison of these structures shows that during head maturation, in addition to capsid expansion, the portal protein undergoes conformational changes to establish interactions with the connector proteins. Additionally, the independently assembled tail undergoes morphological alterations at its proximal end, facilitating its connection to the head-tail joining protein and resulting in the formation of a stable portal-connector-tail complex. The B-DNA molecule spirally glides through the tube, interacting with the nozzle blade region of the middle-ring connector protein. These insights elucidate a mechanism for portal maturation and DNA translocation within the phage lambda system.
OBJECTIVE: The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.

Artificial biomolecular condensates are emerging as a versatile approach to organize molecular targets and reactions without the need for lipid membranes. Here we ask whether the temporal response of artificial condensates can be controlled via designed chemical reactions. We address this general question by considering a model problem in which a phase separating component participates in reactions that dynamically activate or deactivate its ability to self-attract. Through a theoretical model we illustrate the transient and equilibrium effects of reactions, linking condensate response and reaction parameters. We experimentally realize our model problem using star-shaped DNA motifs known as nanostars to generate condensates, and we take advantage of strand invasion and displacement reactions to kinetically control the capacity of nanostars to interact. We demonstrate reversible dissolution and growth of DNA condensates in the presence of specific DNA inputs, and we characterize the role of toehold domains, nanostar size, and nanostar valency. Our results will support the development of artificial biomolecular condensates that can adapt to environmental changes with prescribed temporal dynamics.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific \"helper\" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the novel dynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show that in the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. We show that pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)2 tetramers from spermatid DNA after the removal of H2A-H2B dimers. Our study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis.

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific \"helper\" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal in solution and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.