Polymerase Translocation Research Articles

A topological model for chromatin transcription and a role for nucleosome linkers

There is a good deal of evidence that transcribing RNA polymerase may translocate across nucleosomes without their displacement and (or) rearrangement. A topological model for RNA chain elongation on a nucleosome is considered here. A new mechanism of RNA polymerase translocation is suggested in order to avoid the steric hindrances inherent in the model. It is shown that a transcribed nucleoprotein fiber should be interrupted by protein-free DNA stretches (nucleosome linkers) to allow release of nascent RNA. Possible verificiations and consequences of the model are discussed.

Ordered sequential mechanism of substrate recognition and binding by KB cell DNA polymerase alpha.

We have used a steady-state kinetic approach in conjunction with direct velocity gradient sedimentation binding studies to examine the detailed steps that are involved in the recognition of DNA primer-template and dNTPs by near-homogeneous human DNA polymerase alpha. We demonstrate that the interaction of the polymerase with its substrates obeys a rigidly ordered sequential terreactant mechanism, with template as the first substrate, followed by primer as the second substrate and dNTP as the third. Although the binding of primer is prerequisite to the kinetically significant binding of dNTP, specification of which of the four dNTPs can then add to the enzyme is absolutely determined by base sequence of the template (the first substrate). The critical element in the proof of the ordered mechanism is the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically com the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically com the demonstration of the phenomenon of induced substrate inhibition; the presence of a dideoxy-terminated primer (dead-end inhibitor) induces substrate inhibition by dNTP which is absolutely restricted to the dNTP complementary to the template to which the blocked primer is annealed. This inhibition is kinetically competitive with 3' -hydroxyl-terminated (unblocked) primer and approaches 100% at saturating levels of the complementary dNTP. Direct binding studies document the specific and exclusive ability of complementary dNTPs to drive the polymerase into a stable dead-end complex with the proposed structure, enzyme.template.dideoxy primer.dNTP, thus corroborating the kinetic observations. Attempts to elucidate the order of product release from the enzyme by product inhibition studies have shown the polymerization reaction to be essentially irreversible and have thus been unsuccessful. On the basis the known processivity of KB cell DNA polymerase alpha, a preliminary model involving initial release of pyrophosphate is reasonable; however, the relationship between product release and the process of polymerase translocation remains obscure. All of the kinetic and sedimentation binding studies were performed on a variety of homopolymeric and natural heteropolymeric DNA substrates, and the consistency of the results establishes absolutely the qualitative identity of the general mechanism by which human DNA polymerase alpha recognizes and replicated polydeoxynucleotide primer-templates, regardless of their precise physicochemical nature.

Psychiatric Presentations of Medical Illness: Somatopsychic Disorders

<h3>Abstract</h3> In <i>Escherichia coli</i>, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we develop a spatially resolved supercoiling model of transcription, describing RNAP-supercoiling interactions, topoisomerase activities, stochastic topological domain formation, and supercoiling diffusion in all transcription stages. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes. It reveals that supercoiling transmits RNAP-accessible information through DNA and enables different RNAP molecules to communicate within and between genes. It thus predicts that a topological domain could serve as a transcription regulator, generating substantial transcription bursting and coordinating communications between adjacent genes in the domain. The model provides a quantitative platform for further theoretical and experimental investigations of how genome organization impacts transcription. <h3>Author Summary</h3> DNA mechanics and transcription dynamics are intimately coupled. During transcription, the translocation of RNA polymerase overwinds the DNA ahead and underwinds the DNA behind, rendering the DNA supercoiled. The supercoiled DNA could, in return, influences the behavior of the RNA polymerase, and consequently the amount of mRNA product it makes. Furthermore, supercoils could propagate on the DNA over thousands of base pairs, impacting RNA polymerase molecules at faraway sites. These complicated interplays between supercoiling and RNA polymerase makes supercoiling an important transcription regulator. To quantitatively investigate the role of supercoiling in transcription, we build a spatially resolved model that links transcription with the generation, propagation, and dissipation of supercoiling. Our model reveals that supercoiling mediates transcription at multiple length scales. At a single-gene scale, we show that supercoiling gives rise to the collective motion of co-transcribing RNA polymerase molecules, supporting recent experimental observations. Additionally, large variations in mRNA production of a gene can arise from the constraints of supercoiling diffusion in a topological domain. At a multi-gene scale, we show that supercoiling dynamics allow two adjacent genes influence each other’s transcription kinetics, thus serving as a transcription regulator.

Association of DNA polymerase alpha and beta with rough endoplasmic reticulum in sea-urchin eggs and changes in subcellular distribution during early embryogenesis.

The subcellular distribution of DNA polymerase alpha and beta was examined in unfertilized eggs and embryos of the sea-urchin, Hemicentrotus pulcherrimus. In unfertilized egg homogenates, prepared in sucrose solution containing 5 mM 2-mercaptoethanol and 5 mM MgCl2, DNA polymerases equilibrated in isospycnic centrifugation as a single peak at a buoyant density of 1.261 g/cm3 (band I). This indicates that DNA polymerases associate with a cytoplasmic organelle. Band I was converted to bands II (1.227 g/cm3), III (1.177 g/cm3) and IV (1.146 g/cm3) by EDTA treatment. The RNA content of bands I, II, III and IV was approximately 0.34, 0.25, 0.10 and 0.04 mg/mg protein respectively. In isokinetic and isopycnic centrifugations both DNA polymerase and RNA cosedimented and coequilibrated. These results suggest that bands I, II, III and IV contain various amounts of ribosomes on a common structure. Examination by electron microscopy indicated that bands I, II, III and IV contained mainly monolayered membrane vesicles with different amounts of bound ribosomes. The content of ribosomes varied in the order: band I > band II > band III > band IV. Each band contained DNA polymerase with sedimentation coefficients of 5.8-7.6 S (sensitive to N-ethylmaleimide and aphidicolin) and 3.2 S (insensitive to these drugs). We conclude that almost all of DNA polymerases alpha and beta are localized on the rough endoplasmic reticulum in unfertilized eggs. Mixing experiments suggest that the association is specific and is not an artifact of homogenization. During development of embryos DNA polymerases associated with the membrane decreased with concomitant increase in the nuclear fraction. This suggests that the enzymes migrate from the cytoplasm to the nucleus. It is discussed that the role of the endoplasmic reticulum as a storage site of DNA polymerases in unfertilized eggs and the mechanism of translocation of DNA polymerases from the cytoplasm to the nucleus during early embryogenesis.

RNA chain elongation on a chromatin template.

The rate of RNA chain elongation has been measured with DNA and chromatin as template. RNA propagation on chromatin is about 50% of the rate found with DNA. Kinetic experiments demonstrate that the inhibition is not due to interference with the addition of the nucleoside triphosphates. Analysis of the dependence of propagation on the Tm of DNA shows that the chromatin proteins interfere with the translocation of the RNA polymerase along the DNA template.

Molecular association of DNA polymase with chromatin in sea urchin embryos.

DURING the early cleavage stages of sea urchin embryos, the number of nuclei doubles in 2 h or less and the DNA doubles in about 10–12 min at 16° C1. A special relationship between nuclear DNA and DNA polymerase is suggested by the constant ratio of polymerase activity to DNA2,3. Evidence suggests that this proportionality is maintained by the translocation of the stored polymerase from the cytoplasm to the nucleus4.