The nucleoskeleton and the topology of transcription

Abstract

Transcription is conventionally believed to occur by passage of a mobile polymerase along a fixed template. Evidence for this model is derived almost entirely from material prepared using hypotonic salt concentrations. Studies on subnuclear structures isolated using hypertonic conditions, and more recently using conditions closer to the physiological, suggest an alternative. Transcription occurs as the template moves past a polymerase attached to a nucleoskeleton; this skeleton is the active site of transcription. Evidence for the two models is summarised. Much of it is consistent with the polymerase being attached and not freely diffusible. Some consequences of such a model are discussed.The accepted model for transcription contains three essential participants -the template, polymerase and nascent RNA [I, 21. Transcription is initiated by a diffusible polymerase binding to a promoter; the polymerase then processes along the DNA (Fig. 1 A). As knowledge has increased, other occasional participants have been added (e. g. transcription factors, topoisomerases) but not any other central players. Evidence for this model comes almost entirely from studies using hypotonic salt concentrations, largely because chromatin aggregates in physiological concentrations and so becomes difficult to use. In contrast, studies on preparations isolated using hyper-or iso-tonic salt concentrations suggest that active RNA polymerase is associated with a nucleoskeleton and is not freely diffusible. This nucleoskeleton is seen as the active site of RNA synthesis and transcription occurs by movement of DNA past the attachcd polymerase (Fig. 1 B).Ultimately models will be distinguished by reconstructing efficient transcription in vitro from purified constituents. Proof of the model involving an attached polymerase will require measurement ofits association constant for a skeleton. In the meantime, the two models can be distinguished operationally by determining whether the polymerase is freely diffusible or attached to a larger structure. Much evidence for the 'text-book' model is consistent with the alternative; this alternative has a number of important consequences and these are examined. Discussion centrcs on RNA polymerase 11, the enzyme that transcribes most eukaryotic genes, but applies equally to other polymerases. ARTEFACTSStructure within isolated nuclei cannot be discussed sensibly without some consideration of problems caused by artefacts. They arise because nuclei and chromatin aggregate in physiological salt concentrations 131 ; therefore unphysiological conditions are almost invariably used. ControversyCorrespondence to P. R. Cook, Sir William Dunn School of Pathology, Universlty of Oxford, South Parks Road, Oxford OX1 3RE. UK centres on whether structures seen In vitro are any more than isolation artefacts. RNA, DNA and protein, each concentrated in the nucleus at about 0.1 g/ml, might be expected to aggregate as soon as ion concentrations are altered. (For reviews of the controversy, see [4-61.) Nuclei are usually isol...

Nuclear RNAs confined to a reticular compartment between chromosome territories

“…Both sites of replication and transcription are apparently fixed and remain associated with the structures in nuclei after a nuclear matrix or nucleoskeleton preparation has been performed [18,19]. Replication and transcription patterns appear to be similar but localize to different compartments in the nucleus [20,21].…”

Excision close to matrix attachment regions of the entire chicken alpha-globin gene domain by nuclease S1 and characterization of the framing structures.

“…In the nuclei of eukaryotic cells, the DNA is organized into large loops fixed to the nuclear skeleton (1)(2)(3)(4)(5). The specificity of loop organization-i.e., the specificity of distribution of the loop borders in the genome-has been intensively studied but the experimental results remain controversial (1,4).…”

Crosslinking of nuclear proteins to DNA by cis‐diammminedichloroplatinum in intact cells Involvement of nuclear matrix proteins

“…A possible criticism of these results is that they have been obtained after introducing some modification in the native structure of the nuclei. Many different preparations of the nuclear matrix have been proposed, and possibly no one is immune from drawbacks [9]. Thus, for example, extraction of nuclei with high salt may cause a sliding of DNA over its attachment points or an exchange of the DNA-binding proteins, so that the original DNA-protein interactions are altered or lost.…”