Architecture of the Nuclear Periphery of Rat Pachytene Spermatocytes: Distribution of Nuclear Envelope Proteins in Relation to Synaptonemal Complex Attachment Sites

The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs.

The architecture of the human Rad54–DNA complex provides evidence for protein translocation along DNA

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54–DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.

Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.