The human ribosomal RNA promoter contains two distinct control elements (UCE and core) both of which are recognized by the sequence-specific DNA binding protein UBF1, which has now been purified to apparent homogeneity. The purified factor activates RNA polymerase I (RNA pol I) transcription through direct interactions with either control element. A second RNA pol I transcription factor, designated SL1, participates in the promoter recognition process and is required to reconstitute transcription in vitro. Although SL1 alone has no sequence-specific DNA binding activity, deoxyribonuclease I footprinting experiments reveal that a cooperative interaction between UBF1 and SL1 leads to the formation of a new protein-DNA complex at the UCE and core elements. In vitro transcription experiments indicate that formation of the UBF1-SL1 complex is vital for transcriptional activation by UBF1. Thus, protein-protein interactions between UBF1 and SL1 are required for targeting of SL1 to cis-control sequences of the promoter.

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A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.To understand the mechanism of transcription initiation, it is necessary both to define the cis-acting promoter sequences and to characterize the trans-acting proteins that interact with the promoter to carry out the synthesis of RNA. The development of an extract that efficiently catalyzes the de novo initiation of RNA synthesis in vitro has proved to be very useful not only for mapping promoter elements but also for subsequent biochemical analysis of the transcription machinery. These in vitro studies have revealed that each of the three distinct classes of RNA polymerase in eucaryotes displays its own specificity and mechanism for promoter utilization (30 and references contained therein).The genes encoding the major rRNA species are actively transcribed in the nucleoli of animal cells and account for most of the RNA synthesized in growing cells. Unlike RNA polymerases II and III, RNA polymerase I acts on only one type of promoter sequence that is reiterated about 200 times per haploid mammalian genome. The transcription of rRNA is regulated and responds to a wide range of stimuli, including the growth state of the cell, nutrient starvation, cell cycle regulation, and viral infection (1,10,13,15,22,26,28,29,31). Thus, the synthesis of rRNA by RNA polymerase I offers an opportunity to study a unique regulatory system. Several laboratories used in vitro transcription systems to map the sequences that constitute the promoter region recognized by mammalian RNA polymerase I (11, 17). These studies also revealed an unexpected and interesting specificity intrinsic to rRNA transcription. Extracts that are prepared from human cells and that actively transcribe the human rRNA promoter fail to initiate transcription from the mouse rRNA promoter and vice versa (12, 18). A comparison of the regula...

We (1) provides a sensitive control mechanism that responds to the levels of serum cholesterol and coordinates isoprenoid metabolism to ensure the availability of both sterol and nonsterol products. The plant isoprenoid biosynthetic pathway has numerous additional branches that give rise to a number of unique products, including growth regulators (such as cytokinin, gibberellin, and abscisic acid), photosynthetic pigments, phytotoxins, phytoalexins, and a variety of specialized terpenoids. The regulated synthesis of these isoprenoid compounds is essential to plant growth and development. Studies of HMG-CoA reductase regulation in plants suggest that the enzyme activity responds to a variety of external stimuli, including light (2-4), plant growth regulators (3, 5, 6), sterols (5), and wounding and plant pathogens (7,8). Furthermore, the levels of HMG-CoA reductase activity vary markedly at different stages of development and in different plant tissues (9). Whereas the mammalian and yeast enzymes appear to be localized in the cytoplasm (10, 12), plant HMG-CoA reductase has been found in mitochondria and plastids as well as the microsomal fractions in some, but not all, species (2-8, 13). These studies raise the possibility that independent isoprenoid pathways operate in each of the compartments (14). However, the membrane association of the protein makes these determinations difficult; thus, the studies of plant HMG (20) with modifications. Single-stranded DNA was prepared as described (18) Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; MVA, mevalonic acid. 2779The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.