Transcription of nucleosomal DNA in SV40 minichromosomes by eukaryotic and prokaryotic RNA polymerases

Meneguzzi, Guerríno; Chenciner, Nicole; Milanesi, G.

doi:10.1093/nar/6.8.2947

Cited by 16 publications

(3 citation statements)

References 29 publications

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“…As expected, the rate of incorporation of [3H]UMP with the SV40 minichromosome was approximately 20% of that of naked SV40 DNA. This is in agreement with the results of previous investigations (33). Quite surprisingly, however, we found that the SV40 core was a more efficient template for transcription.…”

Section: Methodssupporting

confidence: 94%

Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions

Brady

Lavialle

Salzman

1980

J Virol

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Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis- N-N′ -tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP 1 and VP 2 in addition to the proteins found associated with the viral minichromosome, i.e., VP 3 and histones H 2 A, H 2 B, H 3 , and H 4 . Our results suggest that the association of VP 1 , VP 2 , or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the “early” SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP 1 or VP 2 or both enhances the transcriptional activity and suggest that these “late” viral proteins may play a role in the regulation of expression of the SV40 genome.

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Section: Methodssupporting

confidence: 94%

Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions

Brady

Lavialle

Salzman

1980

J Virol

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“…This has led a number of investigators to address the question of whether chromatin, and more specifically nucleosome core particles, present an impediment of any sort to transcription in vitro. Early studies using bacterial polymerases (Williamson and Felsenfeld, 1978;Wasylyk et al, 1979) as well as eukaryotic polymerases I and II (Meneguzzi et al, 1979;Wasylyk and Chambon, 1979) suggested that nucleosomes inhibit transcription, in the sense that shorter transcripts were observed from reconstituted than from naked templates, and rates of elongation were decreased. At the same time, transcripts of length corresponding to several nucleosomes were observed, leading to the inference that nucleosomes did not completely impede transcriptional elongation.…”

Section: Introductionmentioning

confidence: 99%

Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro.

Morse

1989

The EMBO Journal

106

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To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core histones and plasmid DNA bearing the Xenopus 5S RNA gene were assembled into nucleosomes and used as templates for transcription in a Xenopus oocyte nuclear extract. Plasmids having a nucleosome incorporating a specific region of the gene were selected by treating the reconstituted molecules with restriction endonucleases. In this way, it was shown that a nucleosome on or close to the internal control region of the 5S RNA gene inhibits transcription. Furthermore, experiments with 5S maxigenes showed that RNA polymerase III, in contrast to SP6 RNA polymerase, will not transcribe through a nucleosome in vitro.

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“…One obvious expectation is that replication (and transcription) should slow down at the sites where the nucleosomes are encountered. Although there is some indirect experimental evidence to this effect (11,21), conclusive results can only be obtained by direct comparison of the positions of the pause sites with the locations of nucleosomes. In the recent work on simian virus 40 (SV40) replication (22), the distribution of the pause sites along the The distribution of the pause sites along the SV40 genome, based on the data obtained by cloning of isolated nascent DNA and subsequent length determinations (22), is presented in Fig.…”

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confidence: 99%

Simian virus 40 replication pause sites map with the nucleosomes

Trifonov¹,

Mengeritsky²

1984

J Virol

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The locations of replication pause sites in the simian virus 40 minichromosome which were determined by sizing cloned fragments of nascent DNA (Zannis-Hadjopoulos et al., J. Mol. Biol. 165:599-607, 1983) were compared with the positions of simian virus 40 nucleosomes in the genome, as obtained by sequence-directed mapping (G. Mengeritsky and E. N. Trifonov, Nucleic Acids Res. 11:3833-3851, 1983; Mengeritsky and Trifonov, Cell Biophys. 6:1-8, 1984). Clear correlation between these two maps is demonstrated, suggesting that nucleosomes hinder propagation of the replication forks.

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Transcription of nucleosomal DNA in SV40 minichromosomes by eukaryotic and prokaryotic RNA polymerases

Cited by 16 publications

References 29 publications

Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions

Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions

Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro.

Simian virus 40 replication pause sites map with the nucleosomes

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