Control region for adenovirus VA RNA transcription.

Abstract

Plasmids containing the VA RNA genes of adenovirus are faithfully transcribed by a crude cytoplasmic extract containing DNA-dependent RNApolymerase II [Wu, G.-J. (1978) Proc. NatI. Acad. Sci. USA 75, 2175USA 75, -2179. By subjecting these DNA templates to in vitro site-directed mutagenesis with a novel enzyme of Pseudomonas and recloning in pBR322, we have constructed an ordered series of deletions which affect the in vitro transcription of the major RNA polymerase III viral product, VA, RNA. Three regions that are required for specific synthesis of VA, RNA can be defined. One, inside the gene at nucleotides + 10 to +76, affects the transcription in an all-or-none fashion. Transcription is initiated on plasmid sequences that replace up to 10 nucleotides downstream from the 5' end ofthe gene. The development of soluble cell-free extracts in which specific genes are selectively and accurately transcribed has greatly facilitated our understanding of the molecular basis of control in prokaryotes (1) and eukaryotes (2)(3)(4)(5)(6). Faithful in vitro transcription systems were first developed for RNA polymerase III products, like the adenovirus encoded 5.5S RNA (VA RNA) by Wu (2) and later modified (4) or the Xenopus 5S genes (3, 7). Appropriate templates in large amounts and with high purity can now be obtained by recombinant DNA techniques. These templates can be manipulated to determine the DNA signals required for control of accurate initiation or termination of transcription.Recently it was shown that, for the 5S genes ofXenopus laevis, a region internal to the gene was required for specific initiation of transcription mediated by oocyte cell extracts (8,9). A purified factor required for accurate initiation strongly bound to this region of the gene (10) and might participate in the temporal control of 5S transcription in vivo because it also binds to the 5S RNA gene product (10,11). A different set of factors seems to be required for adenovirus VA transcription (10) and it was therefore ofinterest to establish whether a similar internal control region could be identified for this RNA (15,16,21).Using the DNA sequence data from this region ofthe genome (17,18,(21)(22)(23) and appropriate restriction enzymes, we have constructed plasmids that allow us to define the DNA signals required for initiation and termination of VA, RNA transcription. We have found an internal control region for VA, transcription initiation located close to the 5' end of the respective gene, as determined by an ordered set of deletions constructed by using the nuclease BAL 31.MATERIALS AND METHODS DNA Templates. Restriction enzyme fragments were prepared from whole adenovirus 2 (Ad2) DNA purified on CsCl density gradients as described (16) and inserted into plasmid pBR322. We constructed a wild-type clone containing the Ad2 sequences from the Sal I site (coordinate 26.9) to the HindIII site (coordinate 31.5) on the physical Ad2 map which goes from 0 to 100. dl 5' +70 (deletion of all sequences upstream from nucleotide +70) was ...

Transcription of two classes of rat growth hormone gene-associated repetitive DNA: differences in activity and effects of tandem repeat structure

“…However, the upstream start site may be positioned by an 11 mer, the GAGAGGGCTGG sequence, that spans the start of the first repeat (Fig. IA) and which has significant homology with the Pol III A box promoter consensus sequence (20,21,(30)(31)(32)47). Alternatively, initiation site selection by Pol III may depend on the 5' flanking sequence, as previously shown for tRNA genes (40,41).…”

“…The human Alu-repeat clones, pPD8 and pPD6, were kindly provided by Drs. T. Friedmann and P. Deininger; adenovirus VA1-5'A55 DNA was a generous gift of Dr. R. Weinmann (5,21). Plasmid DNAs were purified by banding in CsCl gradients (22,23).…”

Adenovirus mutants with DNA sequence perturbations in the intragenic promoter of VAI RNA gene allow the enhanced transcription of VAII RNA gene in HeLa cells

“…These results suggest that an increased quantity of Earlier workers have shown that the VAI gene is a strong competitor for transcription of VAII gene (9,10) presumably competing for initiation factors. Deletion mapping studies demonstrated that only nucleotide sequences from +54 to +69 of VAI promoter are capable of competing for transcription with a second VAI gene in vitro (10). The mechanism of initiation of transcription of class III genes is being investigated intensely in several laboratories.…”

“…the VAI gene competing strongly with the VAII gene for one or more transcriptional factors, thus limiting the transcription of VAII gene (13). In in vitro transcription assays, DNA sequences from +54 to +69 of VAI gene promoter are capable of competing for transcription with another VAI gene or VAII gene (10).…”

“…Using in vitro transcription systems, it was shown that the DNA sequences required for thE transcription of the VAI gene were located within its coding sequence (from +10 to +69 ref. 9,10,11; the numbering here is relative to the starting point of the VAI-G RNA). DNA sequences required for the VAII gene transcription have not been determined; but they are very likely to be in a similar location within the coding sequences of VAII gene.…”