Models for replication and transcription often display polymerases that track like locomotives along their DNA templates. However, recent evidence supports an alternative model in which DNA and RNA polymerases are immobilized by attachment to larger structures, where they reel in their templates and extrude newly made nucleic acids. These polymerases do not act independently; they are concentrated in discrete "factories," where they work together on many different templates. Evidence for models involving tracking and immobile polymerases is reviewed.

HeLa cells were encapsulated in agarose microbeads, permeabilized and incubated with Br‐UTP in a ‘physiological’ buffer; then sites of RNA synthesis were immunolabelled using an antibody that reacts with Br‐RNA. After extending nascent RNA chains by < 400 nucleotides in vitro, approximately 300–500 focal synthetic sites can be seen in each nucleus by fluorescence microscopy. Most foci also contain a component of the splicing apparatus detected by an anti‐Sm antibody. alpha‐amanitin, an inhibitor of RNA polymerase II, prevents incorporation into these foci; then, using a slightly higher salt concentration, approximately 25 nucleolar foci became clearly visible. Both nucleolar and extra‐nucleolar foci remain after nucleolytic removal of approximately 90% chromatin. An underlying structure probably organizes groups of transcription units into ‘factories’ where transcripts are both synthesized and processed.

Molecular dynamics simulations are used to model proteins that diffuse to DNA, bind, and dissociate; in the absence of any explicit interaction between proteins, or between templates, binding spontaneously induces local DNA compaction and protein aggregation. Small bivalent proteins form into rows [as on binding of the bacterial histone-like nucleoid-structuring protein (H-NS)], large proteins into quasi-spherical aggregates (as on nanoparticle binding), and cylinders with eight binding sites (representing octameric nucleosomal cores) into irregularly folded clusters (like those seen in nucleosomal strings). Binding of RNA polymerase II and a transcription factor (NFκB) to the appropriate sites on four human chromosomes generates protein clusters analogous to transcription factories, multiscale loops, and intrachromosomal contacts that mimic those found in vivo. We suggest that this emergent behavior of clustering is driven by an entropic bridging-induced attraction that minimizes bending and looping penalties in the template.polymer physics | Brownian dynamics | chromatin looping | nucleosome D NA in living cells associates with proteins that continuously bind and dissociate. Some proteins affect local structure (such as histones and histone-like proteins), whereas others act globally to compact whole chromosomal segments [such as CCCTC-binding factor (CTCF)] (1-3). Bound proteins also cluster into supramolecular structures; for example, different transcription factors often bind to the same hot spots in the fly genome (4), and active molecules of RNA polymerase II coassociate in transcription factories (5, 6). In the latter case, clustering generates high local concentrations that facilitate production of the appropriate transcripts, as well as organizing the genome in 3D space.Against this background, biophysicists have begun to model DNA folding driven by DNA-binding proteins (3,(7)(8)(9)(10)(11)(12). Usually, the effects of DNA binding are incorporated into an effective potential that influences DNA dynamics; for instance, by stipulating that selected protein-binding regions in the polymer attract each other (11,12). Here, we use molecular dynamics (MD) to model proteins that diffuse to DNA, bind, and dissociate. In the absence of any explicit mutual attraction between proteins or between monomers in the polymer, we uncover an emergent property of the system: binding spontaneously induces protein clustering and genome compaction. For example, simulations yield structures seen experimentally when proteins representing bacterial histone-like nucleoid-structuring protein (H-NS) (1, 2, 13), gold nanoparticles (14, 15), and nucleosome cores bind to DNA. Using data derived from ChIP coupled to high-throughput sequencing (ChIP-seq) (16), we also model binding of RNA polymerase II and its transcription factor, NFκB, to the appropriate (cognate) sites on four human chromosomes; the two proteins spontaneously cluster into factories that are surrounded by loops that reflect those detected in cells using chromatin inte...

No abstract

It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei. We localized translation sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II. This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.

Cellular structures are shaped by hydrogen and ionic bonds, plus van der Waals and hydrophobic forces. In cells crowded with macromolecules, a little-known and distinct force—the “depletion attraction”—also acts. We review evidence that this force assists in the assembly of a wide range of cellular structures, ranging from the cytoskeleton to chromatin loops and whole chromosomes.

Biophysicists are modeling conformations of interphase chromosomes, often basing the strengths of interactions between segments distant on the genetic map on contact frequencies determined experimentally. Here, instead, we develop a fitting-free, minimal model: bivalent or multivalent red and green ‘transcription factors’ bind to cognate sites in strings of beads (‘chromatin’) to form molecular bridges stabilizing loops. In the absence of additional explicit forces, molecular dynamic simulations reveal that bound factors spontaneously cluster—red with red, green with green, but rarely red with green—to give structures reminiscent of transcription factories. Binding of just two transcription factors (or proteins) to active and inactive regions of human chromosomes yields rosettes, topological domains and contact maps much like those seen experimentally. This emergent ‘bridging-induced attraction’ proves to be a robust, simple and generic force able to organize interphase chromosomes at all scales.

Histones H2A and H2B form part of the same nucleosomal structure as H3 and H4. Stable HeLa cell lines expressing histones H2B, H3, and H4 tagged with green fluorescent protein (GFP) were established; the tagged molecules were assembled into nucleosomes. Although H2B-GFP was distributed like DNA, H3-GFP and H4-GFP were concentrated in euchromatin during interphase and in R-bands in mitotic chromosomes. These differences probably result from an unregulated production of tagged histones and differences in exchange. In both single cells and heterokaryons, photobleaching revealed that H2B-GFP exchanged more rapidly than H3-GFP and H4-GFP. About 3% of H2B exchanged within minutes, whereas ∼40% did so slowly (t 1/2 ∼ 130 min). The rapidly exchanging fraction disappeared in 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole and so may represent H2B in transcriptionally active chromatin. The slowly exchanging fraction was probably associated with chromatin domains surrounding active units. H3-GFP and H4-GFP were assembled into chromatin when DNA was replicated, and then >80% remained bound permanently. These results reveal that the inner core of the nucleosome is very stable, whereas H2B on the surface of active nucleosomes exchanges continually.