Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors 1 1Edited by M. Yahiv

Seither, Peter; Iben, Sebastian; Grummt, Ingrid

doi:10.1006/jmbi.1997.1434

Cited by 65 publications

(55 citation statements)

References 43 publications

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“…Interestingly, this domain plays a similar role at mitosis, where it associates topoisomerase I with the nucleolar organizing regions of mitotic chromosomes, a finding previously also indicated by immunohistochemistry (28,29). At both locations (fibrillar centers of the nucleolus and nucleolar organizing regions) topoisomerase I co-localizes with RNA polymerase I, which is believed to form a holo-enzyme together with a variety of other proteins also involved in rRNA transcription (30). Our data suggest that topoisomerase I could be a regular component of this holo-enzyme.…”

Section: Discussionmentioning

confidence: 56%

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Christensen

Barthelmes

Boege

et al. 2002

Journal of Biological Chemistry

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DNA topoisomerase I releases torsion stress created by DNA transcription. In principle, this activity is required in the nucleoplasm for mRNA synthesis and in the nucleoli for rRNA synthesis. Yet, topoisomerase I is mostly a nucleolar protein. Current belief holds that this preference is triggered by the N-terminal domain of the enzyme, which constitutes a nucleolar import signal. Contradicting this view, we show here that nucleolar accumulation of various fragments of topoisomerase I is correlated with their lesser mobility in this compartment and not with the N-terminal domain being intact or present. Therefore, the N-terminal domain is not likely a nucleolar import signal. We show that it rather serves as an adaptor that anchors a subpopulation of topoisomerase I at fibrillar centers of nucleoli and nucleolar organizer regions of mitotic chromosomes. Thus, it provides a steady association of topoisomerase I with the rDNA and with RNA polymerase I, which is maintained in a living cell during the entire cell cycle.DNA topoisomerase I changes the pitch of DNA helices by cutting one DNA strand and allowing rotation of the other (1). One important role for this mechanism is the release of torsion stress created by DNA transcription. Thus, topoisomerase I activity should in principle be required in the nucleoplasm for the synthesis of mRNA, and in the nucleolus for the synthesis of rRNA. However, the latter task seems to dominate. During interphase, most of topoisomerase I is located in the nucleoli and not in the nucleoplasm, although the enzyme interchanges constantly between these two compartments (2). It has been proposed that nucleolar accumulation of topoisomerase I reflects engagement in rDNA-transcription because the rRNA genes are by far the most heavily transcribed genes in the cell. In keeping with this, studies of genomic DNA cleavage by topoisomerase I have invariantly come up with sites in the rDNA (3-5). However, catalytic interactions with rDNA cannot per se account for the accumulation of topoisomerase I in the nucleoli because stabilization of covalent topoisomerase I⅐DNA intermediates by camptothecin immobilizes the enzyme preferentially at nucleoplasmatic sites and only to a much lesser extent in the nucleoli (2). Along this reasoning the question arises of what restricts topoisomerase I in the nucleolus and how is this phenomenon related to rDNA processing. We show here that in a living cell a sub population of topoisomerase I is constantly anchored at the fibrillar centers of the nucleolus or the nucleolar organizer region of mitotic chromosomes and that this is due to an adaptor function of the N-terminal domain of the enzyme. EXPERIMENTAL PROCEDURESCloning and Cell Culture-GFP 1 chimera of human topoisomerase I and of various fragments of it (see Fig. 3A) were stably expressed in the human embryonal kidney cell line 293 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) using the bicistronic expression vector pMC-2P (6) in which the translational initiation of the...

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Section: Discussionmentioning

confidence: 56%

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Christensen

Barthelmes

Boege

et al. 2002

Journal of Biological Chemistry

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“…TIF-IA has been shown to be part of the Pol I 'holoenzyme', the enzyme moiety that is associated with most, if not all, factors required for transcription initiation (Saez-Vasquez and Pikaard, 1997;Seither et al, 1998;Albert et al, 1999). Moreover, TIF-IA has been shown to interact with TIF-IB/SL1 (Miller et al, 2001), which suggests that TIF-IA bridges both protein complexes.…”

Section: Resultsmentioning

confidence: 99%

Multiple interactions between RNA polymerase I, TIF‐IA and TAF _I subunits regulate preinitiation complex assembly at the ribosomal gene promoter

et al. 2002

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In mammals, growth-dependent regulation of rRNA synthesis is brought about by the transcription initiation factor TIF-IA. TIF-IA is associated with a fraction of the TBP-containing factor TIF-IB/SL1 and the initiation-competent form of RNA polymerase I (Pol I). We investigated the mechanisms that down-regulate cellular pre-rRNA synthesis and demonstrate that nutrient starvation, density arrest and protein synthesis inhibitors inactivate TIF-IA and impair the association of TIF-IA with Pol I. Moreover, we used a panel of TIF-IA deletion mutants to map the domains that mediate the interaction of TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids 512-609 interact with two subunits of Pol I, RPA43 and PAF67, whereas a short, conserved motif (LARAK, amino acids 411-415) is required for the association of TIF-IA with TAF I 95 and TAF I 68. The results uncover an interphase for essential protein-protein interactions that facilitate Pol I preinitiation complex formation.

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“…There have been previous reports that RPI might exist as a holoenzyme (34,35,38,39). However, there have been considerable differences in the composition of the purified RPI complexes.…”

Section: Components Of the Dna Repair/replication System Are Associatmentioning

confidence: 99%

“…Recent reports have demonstrated the existence of various forms of RNA polymerase I (38)(39)(40). However, there are some significant differences in the composition of the various preparations which may reflect differences in the mode of isolation or the species from which they were purified.…”

Section: Introductionmentioning

confidence: 99%

“…However, there are some significant differences in the composition of the various preparations which may reflect differences in the mode of isolation or the species from which they were purified. For example, the mammalian polymerase I complex isolated by Seither et al (38) contained the accessory rDNA-specific transcription factor UBF, but was not self-sufficient for promoter-dependent transcription. In contrast, the Xenopus laevis RNA polymerase I complex purified by Albert et al (39) did not contain detectable amounts of UBF, but was able to autonomously initiate transcription in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system

Hannan¹,

Cavanaugh²,

Hempel³

et al. 1999

Nucleic Acids Research

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Traditional models for transcription initiation by RNA polymerase I include a stepwise assembly of basic transcription factors/regulatory proteins on the core promoter to form a preinitiation complex. In contrast, we have identified a preassembled RNA polymerase I (RPI) complex that contains all the factors necessary and sufficient to initiate transcription from the rDNA promoter in vitro. The purified RPI holoenzyme contains the RPI homolog of TFIID, SL-1 and the rDNA transcription terminator factor (TTF-1), but lacks UBF, an activator of rDNA transcription. Certain components of the DNA repair/replication system, including Ku70/80, DNA topoisomerase I and PCNA, are also associated with the RPI complex. We have found that the holoenzyme supported specific transcription and that specific transcription was stimulated by the RPI transcription activator UBF. These results support the hypothesis that a fraction of the RPI exists as a preassembled, transcriptionally competent complex that is readily recruited to the rDNA promoter, i.e. as a holoenzyme, and provide important new insights into the mechanisms governing initiation by RPI.

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Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors 1 1Edited by M. Yahiv

Cited by 65 publications

References 43 publications

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Multiple interactions between RNA polymerase I, TIF‐IA and TAF _I subunits regulate preinitiation complex assembly at the ribosomal gene promoter

Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system

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Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors 1 1Edited by M. Yahiv

Cited by 65 publications

References 43 publications

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

Multiple interactions between RNA polymerase I, TIF‐IA and TAF I subunits regulate preinitiation complex assembly at the ribosomal gene promoter

Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system

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Multiple interactions between RNA polymerase I, TIF‐IA and TAF _I subunits regulate preinitiation complex assembly at the ribosomal gene promoter