Stable transcription complex on a class III gene in a minichromosome.

Abstract

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.Fractionation of soluble transcription systems has indicated that multiple cellular factors in addition to RNA polymerase III are necessary to transcribe class III genes (14,15). Two factors (designated IIIB and IIIC) are necessary for transcription of the tRNA and the adenovirus VA RNA genes in vitro, whereas the 5S genes require these same factors plus a third gene-specific factor (IIIA) (14, 15). Formation of a stable transcription complex in vitro, containing one or more of these factors, is a general feature of these genes (2, 9, 13). The finding that isolated cellular chromatin is associated with all the factors necessary to promote transcription of the endogenous 5S and tRNA genes by purified RNA polymerase III suggests that such complexes may exist in vivo (11,17). To establish whether these chromatin-associated factors are specifically bound in a stable complex on class III genes, we have studied their interaction with a model chromatin template-the simian virus 40 (SV40) minichromosome.Numerous class III genes cloned into SV40 have been found to be transcriptionally active in the infected cell (8,18,19). During infection, SV40 DNA is packaged by the host cell histones into a chromatin structure termed the minichromosome (16). In light of the stable transcription complexes observed on class III genes in vitro (2, 9, 13) and the association of factor with cellular chromatin (11, 17), we decided to investigate whether the transcription factors that interact with these genes remain bound to the template in a stable complex during the isolation of recombinant minichromosomes containing such genes. Studies were conducted with a Xenopus laevis tRNAMet gene and a Xenopus borealis 5S RNA gene cloned into the late region of SV40. Previous work has shown that formation of a stable complex in vitro requires factors B and C for the tRNAMet gene and factors A and C for the 5S gene (9). Here we show that the tRNAMet gene in the minichromosome can be isolated in a stable complex with factors B and C and is an active in vitro template for purified RNA transcription by purified RNA polymerase III in either the absence or the presence of other factors. MATERIALS AND METHODSVirus construction. The tRNA SV40 fragments, orientations 1 and 2, were constructed as follows. An EcoRI-linkered, 3,055-base-pair fragment of wild-type SV40, extending from the BamHI site to the HpaII site, was cloned into the Ec...

Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates.

“…Thus we may conclude that RNA polymerase II preinitiation complexes resist nucleosome assembly and not simply incubation in Xenopus extracts. The results reported here and elsewhere (17,22) are also in agreement with earlier studies of the transcription of chromatin templates by RNA polymerase III, which demonstrated that the presence of stable transcription complexes on the template allows the subsequent assembly of nucleosomes without loss of transcriptional activity (10,13). In those cases, the complex needed to rescue the template from inactivation by nucleosomes consisted only of certain initiation factors bound to the DNA.…”

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

“…1 and 8]) leads us to conclude that the phased nucleosome observed over the 5S RNA gene on the 2-h minichromosomes is probably responsible for making these minichromosomes refractory to transcriptional activation. Our results are consistent with the work of Lassar et al (37), showing that this 5S RNA gene in a simian virus 40 minichromosome is refractory to transcription by RNA polymerase III in the absence or presence of the other transcription factors. The repression they observed also appeared to be independent of histone Hi, because the block was not relieved by sedimenting the minichromosomes through 0.6 M NaCl.…”

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo.

“…Transcription of the Xenopus laevis oocyte 5S RNA gene is repressed by histones (11,22,71), and histone Hi apparently plays an instrumental role in the in vivo repression of oocyte-type 5S RNA genes (51,73). The 5S gene can also be inhibited by assembly into nucleosomes without histone Hi (4,5,32,38,54,70 (32,73). Similarly, transcription of tRNA genes (and of rRNA genes) is not affected by partial loss of nucleosomes in yeast cells under conditions in which several class II genes are transcriptionally activated (24).…”