Assembly of alternative multiprotein complexes directs rRNA promoter selectivity.

Abstract

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SLI can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.[Key Words: RNA pol I; rRNA transcription; species specificity; transcription factors; UBF; SL1] Received February 7, 1990; revised version accepted March 14, 1990.Transcriptional initiation in eukaryotic cells is a highly regulated process requiring the correct association of numerous proteins into a specific complex with RNA polymerase {Mitchell and Tjian 1989; Saltzman and Weinmann 1989). Although mapping of essential promoter elements has identified multiple DNA sequences important for template recognition, how the different transcription factors work together with RNA polymerase to select these sequences as sites of initiation is poorly understood. Current results suggest strongly that the DNA sequence specificity of a single protein cannot account for promoter recognition by any of the three cellular RNA polymerases {Yoshinaga et al. 1987; Murphy et al. 1989; Smale and Baltimore 19891.Instead, it seems that the interactions of multiple proteins, both with the DNA and with one another, are required to generate the observed selectivity of initiation.The specificity of RNA polymerase I (RNA pol I} transcription is well suited for studies of promoter recognition. Unlike RNA pol II and RNA pol III, which recognize a wide variety of promoters, RNA pol I apparently initiates from only a single type of promoter in the cell, that of the large rRNA gene . This limited promoter range is reflected in the stringent species specificity of RNA pol I transcription.Studies using either intact cells or in vitro transcription extracts indicate that whereas very closely related species [e.g., mouse and rat)...

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

“…An alternative explanation for the ChEC pattern observed would be the presence of two binding sites for this complex at the rDNA promoter. The interaction of a CF component with 5Ј regulatory sequences provides a strong argument for the functional equivalence of yeast CF and the selectivity factor (SL1) in higher eukaryotes, for which such an interaction has been shown in in vitro footprinting experiments (Bell et al 1988(Bell et al , 1990). …”

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids

“…These include Xenopus and mouse enhancers and ribosomal gene promoter domains from Xenopus, human, rat and mouse (discussed in 12, 37). Nonetheless, UBF from all vertebrate species tested produces essentially identical footprints on various promoter or enhancer probes suggesting that vertebrate UBFs recognize DNA in the same way (7,(12)(13)(14)33). To reconcile these observations it has been suggested that the structure of ribosomal gene promoters may have been conserved in evolution without primary sequence conservation (12).…”

“…Human SLI requires interaction with UBF to form a promoter complex which can be visualized as a DNase I footprint that is more extensive than the footprint produced by UBF (33). Therefore, UBF appears to be the primary DNA binding transcription factor in the human system (10,13). In mice, however, UBF is not required for SLI to bind the promoter (34,35) but both factors still interact to form a footprint more extensive than either activity alone (13).…”

“…Therefore, UBF appears to be the primary DNA binding transcription factor in the human system (10,13). In mice, however, UBF is not required for SLI to bind the promoter (34,35) but both factors still interact to form a footprint more extensive than either activity alone (13). Furthermore, several studies have revealed that mouse rRNA transcription is greatly *To whom correspondence should be addressed Q--n) 1994 Oxford University Press stimulated by UBF apparently by acting early in the assembly of stable pre-initiation complexes (10,15,18,34).…”

“…UBF was originally purified by two independent means, first as a human cell fraction necessary for RNA polymerase I transcription in vitro and as an rRNA gene enhancer binding protein (10)(11)(12). UBF has now been purified and cloned from human, frog, rat and mouse (7,(13)(14)(15)(16)(17)(18)(19)(20). The UBF protein consists of an amino terminal dimerization domain (19, 21,22) followed by five repeated 80 amino acid domains (17) termed HMG-boxes due to their similarity to the DNA binding domains of High Mobility Group (HMG) proteins 1 and 2 (16), and a highly acidic carboxyl terminal tail.…”