Molecular BioSystems

doi: 10.1039/b817146gpmid: N/A

doi: 10.1039/b817149cpmid: N/A

doi: 10.1039/b815439bpmid: 18931779

Miele, Adriana; Dekker, Job

doi: 10.1039/b803580fpmid: 18931780

Over the last few years important new insights into the process of long-range gene regulation have been obtained. Gene regulatory elements are found to engage in direct physical interactions with distant target genes and with loci on other chromosomes to modulate transcription. An overview of recently discovered long-range chromosomal interactions is presented, and a network approach is proposed to unravel gene-element relationships.Gene expression is controlled by regulatory elements that can be located far away along the chromosome or in some cases even on other chromosomes. Genes and regulatory elements physically associate with each other resulting in complex genome-wide networks of chromosomal interactions. Here we describe several well-characterized cases of long-range interactions involved in the activation and repression of transcription. We speculate on how these interactions may affect gene expression and outline possible mechanisms that may facilitate encounters between distant elements. Finally, we propose that a genome-wide network analysis may provide new insights into the logic of long-range gene regulation.

Clore, G. Marius

doi: 10.1039/b810232epmid: 18931781

Many biological macromolecular interactions proceed via lowly-populated, highly transient species that arise from rare excursions between the minimum free energy configuration and other local minima of the free energy landscape. Little is known about the structural properties of such lowly-occupied states since they are difficult to trap and hence inaccessible to conventional structural and biophysical techniques. Yet these states play a crucial role in a variety of dynamical processes including molecular recognition and binding, allostery, induced-fit and self-assembly. Here we highlight recent progress in paramagnetic nuclear magnetic resonance to detect, visualize and characterize lowly-populated transient species at equilibrium. The underlying principle involves the application of paramagnetic relaxation enhancement (PRE) in the fast exchange regime. Under these conditions the footprint of the minor species can be observed in the PRE profiles measured for the major species, providing distances between the paramagnetic label and protons of interest are shorter in the minor species than the major one. Ensemble simulated annealing refinement directly against the PRE data permits one to obtain structural data on the minor species. We have used the PRE (a) to detect and characterize the stochastic target search process whereby a sequence-specific transcription factor (the Hox-D9 homeodomain) binds to non-cognate DNA sites as a means of enhancing the rate of specific association via intramolecular sliding and intermolecular translocation; (b) to directly visualize the distribution of non-specific transient encounter complexes involved in the formation of stereospecific protein–protein complexes; (c) to detect and visualize ultra-weak self-association of a protein, a process that is relevant to early nucleation events involved in the formation of higher order structures; and (d) to determine the structure of a minor species for a multidomain protein (maltose binding protein) where large interdomain motions are associated with ligand binding, thereby shedding direct light on the fundamental question of allostery versus induced fit in this system. The PRE offers unique opportunities to directly probe and explore in structural terms lowly-populated regions of the free energy landscape and promises to yield fundamental new insights into biophysical processes.

Spiering, Michelle M.; Nelson, Scott W.; Benkovic, Stephen J.

doi: 10.1039/b812163jpmid: 18931782

Our studies on the T4 replisome build on the seminal work from the Alberts laboratory. They discovered essentially all the proteins that constitute the T4 replisome, isolated them, and measured their enzymatic activities. Ultimately, in brilliant experiments they reconstituted in vitro a functioning replisome and in the absence of structural information created a mosaic as to how such a machine might be assembled. Their consideration of the problem of continuous leading strand synthesis opposing discontinuous lagging strand synthesis led to their imaginative proposal of the trombone model, an illustration that graces all textbooks of biochemistry. Our subsequent work deepens their findings through experiments that focus on defining the kinetics, structural elements, and protein–protein contacts essential for replisome assembly and function. In this highlight we address when Okazaki primer synthesis is initiated and how the primer is captured by a recycling lagging strand polymerase—problems that the Alberts laboratory likewise found mysterious and significant for all replisomes.

Yao, Nina Y.; O’Donnell, Mike

doi: 10.1039/b811097bpmid: 18931783

Replisomes are dynamic multiprotein machines capable of simultaneously replicating both strands of the DNA duplex. This review focuses on the structure and function of the E. coli replisome, many features of which generalize to other bacteria and eukaryotic cells. For example, the bacterial replisome utilizes clamps and clamp loaders to coordinate the actions required of the trombone model of lagging strand synthesis made famous by Bruce Alberts. All cells contain clamps and clamp loaders and this review summarizes their structure and function. Clamp loaders are pentameric spirals that bind DNA in a structure specific fashion and thread it through the ring shaped clamp. The recent structure of the E. coli β clamp in complex with primed DNA has implications for how multiple polymerases function on sliding clamps and how the primed DNA template is exchanged between them. Recent studies reveal a remarkable fluidity in replisome function that enables it to bypass template lesions on either DNA strand. During these processes the polymerases within the replisome functionally uncouple from one another. Mechanistic processes that underlie these actions may involve DNA looping, similar to the trombone loops that mediate the lagging strand Okazaki fragment synthesis cycle.

Formosa, Tim

doi: 10.1039/b812136bpmid: 18931784

FACT is an essential component of the machinery used by eukaryotic cells both to establish and to overcome the nucleosomal barrier to DNA accessibility, and it does so without hydrolyzing ATP. FACT is a transcription elongation factor, but this review stresses additional roles in DNA replication and initiation of transcription. The widely-held model that FACT functions by displacing an H2A–H2B dimer from a nucleosome is examined, and an alternative proposal is presented in which dimer loss can occur but is a secondary effect of a primary structural change induced by FACT binding which we have called “nucleosome reorganization.” The structures of two domains of FACT have been determined and they reveal multiple potential interaction sites. Roles for these binding sites in FACT function and regulation are discussed.

Finkelstein, Ilya J.; Greene, Eric C.

doi: 10.1039/b811681bpmid: 18931785

Single molecule methods offer an unprecedented opportunity to examine complex macromolecular reactions that are obfuscated by ensemble averaging. The application of single molecule techniques to study DNA processing enzymes has revealed new mechanistic details that are unobtainable from bulk biochemical studies. Homologous DNA recombination is a multi-step pathway that is facilitated by numerous enzymes that must precisely and rapidly manipulate diverse DNA substrates to repair potentially lethal breaks in the DNA duplex. In this review, we present an overview of single molecule assays that have been developed to study key aspects of homologous recombination and discuss the unique information gleaned from these experiments.

Minamino, Tohru; Imada, Katsumi; Namba, Keiichi

doi: 10.1039/b808065hpmid: 18931786

Flagellar type III protein export is highly organized and well controlled in a timely manner by dynamic, specific and cooperative interactions among components of the export apparatus, allowing the huge and complex macromolecular assembly to be built efficiently.The bacterial flagellum, which is required for motility, consists of a rotary motor, a universal joint and a helical propeller. Most of the flagellar components are translocated to the distal, growing end of the flagellum for assembly through the central channel of the flagellum itself by the flagellar type III protein export apparatus, which is postulated to be located on the cytoplasmic side of the flagellar basal body. The export specificity switching machinery, which consists of at least two proteins that function as a molecular ruler and an export switch, respectively, monitors the state of hook–basal body assembly in the cell exterior and switches export specificity, thereby coupling sequential flagellar gene expression with the flagellar assembly process. The export ATPase complex composed of an ATPase and its regulator acts as a pilot to deliver its export substrate to the export gate and helps initial entry of the substrate N-terminal chain into a narrow pore of the export gate. The energy of ATP hydrolysis appears to be used to disassemble and release the ATPase complex from the protein about to be exported, and the rest of the successive unfolding/translocation process of the long polypeptide chain is driven solely by proton motive force (PMF), perhaps through biased one-dimensional Brownian diffusion. Interestingly, the subunits of the ATPase complex have significant sequence similarities to subunits of F0F1-ATP synthase, a rotary motor that drives the chemical reaction of ATP synthesis using PMF, and the entire crystal structure of the export ATPase is extremely similar to the α/β subunits of F0F1-ATP synthase, suggesting that the flagellar export apparatus and F0F1-ATP synthase share the mechanism for their two distinct functions.