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ScienceWeek
CELL BIOLOGY: ON NUCLEAR PORES
The following points are made by R. Wozniak and P.R. Clarke (Current Biology 2003 13:R970):
1) The chromatin in every eukaryotic cell is encapsulated in the nucleus by the nuclear envelope, which physically restricts access of molecules to the genome, thereby governing gene expression and DNA replication. To enter the nucleus, all molecules must travel through elaborate macromolecular gateways termed "nuclear pore complexes" (NPCs), which fenestrate the double membrane of the nuclear envelope. The structure of the NPC is largely conserved throughout all eukaryotes and is made up of approximately 30 different proteins, the nucleoporins, with at least eight copies of each protein per NPC [1,2]. It is believed that groups of nucleoporins associate into distinct but interacting subcomplexes which are repeated to give the NPC its characteristic eight-fold symmetry.
2) How an NPC is built from nucleoporins has been a question of intense interest for many years. The assembly of nucleoporins into substructures, which together form the NPC, must be coordinated with changes in membrane structure to produce a trans-cisternal channel through the nuclear envelope in which the NPC is positioned. This process probably involves integral membrane proteins that become part of the NPC. Moreover, NPCs are intimately linked to chromatin and interaction with protein networks such as the nuclear lamina that underlie the nuclear envelope in metazoan cells. The last year has seen advances in understanding the molecular mechanisms of NPC formation including the identification of early assembly intermediates [3-5] and new evidence for the role of nuclear transport factors in this process.
3) In metazoan cells, NPC formation is timed to occur at two points in the cell cycle, during S phase and near the end of mitosis. During S phase, there is a doubling of the total number of NPCs -- while this process is probably coordinated with new membrane synthesis, the NPCs appear to be assembled across the intact nuclear envelope membrane. Later, during prometaphase, NPCs are disassembled and then rebuilt again from their disassembled components during telophase, as the nuclear envelope reforms around the daughter chromatin.
4) This late mitotic assembly process has been the more extensively studied, largely because of the availability of in vitro assays using Xenopus egg extracts containing soluble proteins and membrane vesicle fractions which together are capable of reconstituting nuclear envelope formation around chromatin. Use of this assay system has led to the identification of distinct intermediates in NPC formation and revealed the roles of some individual nucleoporins in this process. Recently, the use of small interfering RNAs (siRNAs) to deplete levels of proteins in tissue culture cells has also emerged as a powerful tool for evaluating the role of individual nucleoporins in the assembly and stability of NPCs.
References (abridged):
1. Rout, M.P., Aitchison, J.D., Suprapto, A., Hjertaas, K., Zhao, Y., and Chait, B.T. (2000). The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635-651
2. Cronshaw, J.M., Krutchinsky, A.N., Zhang, W., Chait, B.T., and Matunis, M.J. (2002). Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915-927
3. Boehmer, T., Enninga, J., Dales, S., Blobel, G., and Zhong, H. (2003). Depletion of a single nucleoporin, Nup107, prevents the assembly of a subset of nucleoporins into the nuclear pore complex. Proc. Natl. Acad. Sci. USA 100, 981-985
4. Walther, T.C., Alves, A., Pickersgill, H., Loiodice, I., Hetzer, M., Galy, V., Hulsmann, B.B., Kocher, T., Wilm, M., Allen, T., Mattaj, I.W., and Doye, V. (2003). The conserved Nup107-160 complex is critical for nuclear pore complex assembly. Cell 113, 195-206
5. Harel, A., Orjalo, A.V., Vincent, T., Lachish-Zalait, A., Vasu, S., Shah, S., Zimmerman, E., Elbaum, M., and Forbes, D.J. (2003). Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores. Mol. Cell. 11, 853-864
Current Biology http://www.current-biology.com
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CELL BIOLOGY: ON THE STRUCTURE OF THE CELL NUCLEUS
The following points are made by A.I. Lamond and J.E. Sleeman (Current Biology 2003 13:R825):
1) The nucleus is the defining feature of eukaryotic cells. It contains the chromosomes and is a site of major metabolic activities, such as DNA replication, gene transcription, RNA processing, and ribosome subunit maturation and assembly. The nucleus is separated from the surrounding cytoplasm by a double membrane. The outer nuclear membrane is continuous with the endoplasmic reticulum, or "rough ER", where the translation of secreted and membrane bound proteins takes place. Thus, the nucleus serves to partition the sites of gene transcription from those of protein synthesis in eukaryotic cells.
2) Movement of proteins and RNA-protein complexes between the nucleus and cytoplasm occurs continually. mRNAs and newly assembled ribosomal subunits are exported from their sites of synthesis in the nucleus to the cytoplasm. Conversely, as all proteins are synthesized in the cytoplasm, nuclear factors, such as histones, transcriptional regulators and splicing factors, are selectively imported into the nucleus. Some proteins, including hnRNP proteins and transport receptors, shuttle repeatedly between the nucleus and cytoplasm. All of this nucleo-cytoplasmic exchange occurs via dedicated multiprotein structures located in the nuclear envelope, termed 'Nuclear Pore Complexes' (NPCs).
3) Like the cytoplasm, the nucleus is compartmentalized. As well as the chromosomes, the nucleoplasm contains numerous classes of "nuclear bodies", including nucleoli, Cajal bodies (CBs), gems, splicing speckles and promyelocytic leukemia (PML) bodies. However, in contrast with cytoplasmic compartments, such as mitochondria, lysosomes, and the Golgi apparatus, the subnuclear compartments lack a membrane separating them from the surrounding nucleoplasm. The accumulation of nuclear factors in distinct nuclear bodies may help to generate a high local concentration of components and, thus, enhance the efficiency of reactions. Furthermore, nuclear compartimentalization may modulate access of enzymes and receptors to their substrates, or physically separate distinct pools of mature/immature and active/inactive factors. Consequently, specific pathways and mechanisms must operate within the nucleus to control the assembly of nuclear bodies and to target factors to the proper locations.
4) Much of the nuclear volume is occupied by the genome, which is packaged into separate chromosomes. Chromosomes are built up through folding and compaction of the DNA-histone complexes. The nuclear DNA-protein complex is termed "chromatin" and contains a host of additional DNA binding and regulatory factors. Nucleosomes are twisted into a helical 10-nm fiber, which is further folded into a 30-nm fiber and perhaps into even higher order structures. This high degree of folding is required in order to fit the large quantity of genomic DNA within the volume of the nucleus. For example, a typical human fibroblast cell must accommodate ~6 x 10^(9) base pairs of DNA within a nuclear volume of ~500 cubic microns(1-5).
References (abridged):
1. Carmo-Fonseca, M. (2002). The contribution of nuclear compartmentalization to gene regulation. Cell 108, 513-521
2. Lamond, A.I. and Spector, D.L. (2003). Nuclear speckles: A model for nuclear organelles. Nat. Rev. Mol. Cell Biol. 4, 605-612
3. Misteli, T. (2001). Protein dynamics: implications for nuclear architecture and gene expression. Science 291, 843-847
4. Ogg, S.C. and Lamond, A.I. (2002). Cajal bodies and coilin -moving towards function. J. Cell Biol. 159, 17-21
5. Spector, D.L. (2003). The dynamics of chromosome organization and gene regulation. Annu. Rev. Biochem. 72, 573-608
Current Biology http://www.current-biology.com
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GENOME BIOLOGY: STRUCTURE OF DNA IN THE NUCLEOSOME
The following points are made by T.J. Richmond and C.A. Davey (Nature 2003 423:145):
1) DNA in eukaryotic cells is packaged repetitively into nucleosomes by means of extensive association with histone proteins. The hierarchical chromatin structure formed is the genomic substrate relevant to the vital processes of DNA replication, recombination, transcription, repair, and chromosome segregation, and to the pathological progression of cancer and viral disease. Although nucleosomal organization of DNA is essentially ubiquitous throughout genomes and generally repressive to gene expression, it also contributes to gene transcription in a gene-specific manner, suggesting that nucleosome positioning in gene promoter regions is important for genuine gene regulation in vivo. The question therefore arises of how chromatin structure, in which DNA is normally highly compacted, permits site-specific access to regulatory factors and more extensive exposure to the transcription apparatus.
2) The answer is likely to require a knowledge of DNA conformation in the nucleosome core. The core comprises 147 base pairs (bp) of DNA and the histone octamer; compared with the nucleosome, it lacks only 10 90 bp of linker DNA envisaged to be naked or bound to histone H1. The histone-fold domains of the octamer organize the central 129 of 147 bp in 1.59 left-handed superhelical turns with a diameter only fourfold that of the double helix. The relatively straight 9-bp terminal segments contribute little to the curvature of the complete 1.67-turn superhelix. So far, the site-specific regulatory factors that have been discovered bind the linker or terminal regions of the intact nucleosome. The lack of binding to the central region of the superhelix might simply be a consequence of bending the double helix or, additionally, of unusual DNA conformations induced by histone binding. At least one protein, HIV-1 integrase, does prefer DNA bent around the nucleosome in contrast to naked DNA.
3) The initiation of DNA-dependent nuclear processes in the context of chromatin implies that nucleosome position is biased by the DNA sequence to facilitate access by initiation factors. Numerous examples of positioned nucleosomes in gene promoter regions have been described both in vivo and in vitro. Preferential positioning could place factor-binding sequences in nucleosome linker or terminal region DNA. Furthermore, nucleosomes are intrinsically mobile and yield access to their DNA in vitro, allowing even RNA polymerase to transcribe nucleosomal DNA without causing dissociation of the histone octamer. In vivo, energy-dependent chromatin remodeling factors, targeted by gene regulatory proteins and acting directly on the nucleosome core, augment nucleosome mobility. Their mechanism of action most probably derives from the innate ability of nucleosomes to "slide" along DNA without releasing it.
4) In summary: The 1.9-angstrom-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fiber formation and chromatin remodeling.
Nature http://www.nature.com/nature
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