Distinctive sequence organization and functional programming of an Alu repeat promoter.

Abstract

Plasmid clones containing a human Alu family repeat can be transcribed efficiently by RNA polymerase mI in HeLa cell extract. This generated three RNA species, all of which initiated from the first base (+1) of the repeat. By studying the transcriptional properties of deletion clones, subclones, and topologically different DNA templates, we demonstrated that: (i) supercoiled DNA templates are transcribed 3-to 5-fold more efficiently than are linear or nicked circular DNA molecules; (ii) a contiguous DNA helix in the transcription complexes that extends into the 5' flanking region of positions -30 to -85 is absolutely required for initiation to occur (this interaction does not involve recognition of specific DNA sequences); and (Mi) similar to the adenovirus VAI RNA and tRNA genes, the Alu repeat 3' to the al-globin gene (designated 3'-al Alu) contains a split intragenic promoter: an anterior element (positions +4 to +37) and a posterior element (positions +70 to +82). However, the promoter of the Alu repeat functions in distinctive ways in comparison to those of other RNA polymerase f-dependent genes. The posterior promoter element alone is sufficient and necessary for an accurate initiation to occur. The presence of the anterior promoter element, which by itself does not initiate transcription, enhances the transcriptional efficiency by a factor of 10-to 20-fold. Furthermore, the distance between the initiation sites and the posterior promoter element, but not the anterior promoter element, remains constant. These results suggest that the promoter of this Alu family repeat consists of at least two functionally different domains: a "directing element" (the posterior promoter element) that determines the accuracy of initiation and an "enhancing element" (the anterior promoter element) that is mainly responsible for the transcriptional efficiency.In eukaryotic cells, RNA polymerase III is responsible for the transcription of genes coding for 5S rRNA, tRNA, and several small viral RNAs, including the adenovirus VAI RNA (for references, see refs. 1-3). The biological roles of 5S rRNA and tRNA in protein synthesis are well documented, whereas the VAI RNA is required for the efficient translation of late viral mRNA (4).The development of oocyte injection techniques (5) and cell-free transcription systems (6-8) has greatly aided the identification of promoter sequences and protein factors involved in the expression of these RNA polymerase II1-dependent genes (class III genes). One novel feature of the transcriptional control regions of class III genes is their location within the coding sequences (intragenic promoter). For example, a contiguous DNA block (from position +50 to +83) is essential for accurate and efficient transcription of Xenopus 5S rRNA genes (9, 10). A 37-kDa protein factor has been purified and shown to bind to the intragenic promoter, a process necessary for the expression of the 5S rRNA gene (11)(12)(13). Similar to 5S rRNA gene, the promoter for transcription of VAI RNA gene is also mapped ...

DNA methylation in the Alu sequences of diploid and haploid primary human cells.

“…The site of in vitro transcriptional initiation by polymerase I1 immediately upstream of the A box had been mapped previously in the Alu element associated with the a1 globin gene (Perez-Stable et al, 1984). These data were confirmed by using as S1 protection probe a 109 bp TaqI-HincH fragment which was labeled at the TaqI site located in the Alu element (map position 12 547).…”

“…Most Alu elements are permanently silenced in living cells (Deininger, 1989), since it would be problematic for a cell to transcribe 5 % of its genome. In cell-free nuclear extracts, many Alu sequences can be efficiently transcribed in vitro by the DNA-dependent RNA polymerase III (Perez-Stable et al, 1984;Perez-Stable and Shen, 1986;Deininger, 1989).…”

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

“…Dot-blot hybridization. [ 32 P]-labeled Alu-containing DNA probe was generated from the clone BLUR 8, 46 by random priming assay. Cellular mRNAs were isolated from either control A431 cells or cells treated with purified proteasomes (to increase the intra-cellular level of proteasome-associated endo-RNAse subunits of the 26S complex (e.g., members of the 19S sub-complex), which were not examined in this study, may also contribute to the RNAse specificity of proteasomes.…”

Regulation of mammalian gene expression by retroelements and non‐coding tandem repeats

“…(31) This promoter contains an anterior element (called the A-box) and a posterior element (called the B-box), which are sufficient for correct transcription initiation. (32) The B-box of tRNA genes and SINEs binds the transcription complex TFIIIC, which can be fractionated into two components, TFIIIC1 and TFIIIC2. (33) Their cooperative interaction dramatically enhances the binding of TFIIIC2 to the B-box.…”