Characterization of factors that direct transcription of rat ribosomal DNA.

Abstract

The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+ 1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.Eucaryotic rRNA genes (rDNAs) code for three of the four rRNA molecules (18S, 5.8S, and 28S rRNAs). These three RNAs are products of degradative processing of a larger precursor (40S to 47S pre-rRNA). The genes that serve as the template for this transcription are present in multiple copies (approximately 200 per haploid genome) and are organized as clusters of tandem repeats. In interphase cells, the genes are localized to the nucleolus, where they are transcribed by RNA polymerase I. Each repeat consists of a transcribed portion and a nontranscribed spacer (reviewed in references 27 and 28). In at least two species, Xenopus laevis and Drosophila melanogaster, the nontranscribed spacer is transcribed (9,23,41), and at least part of the nontranscribed spacer of the rat repeat is transcribed from a spacer promoter (6), suggesting that the name of this region needs to be changed.The transcription initiation sites of several mammalian rRNA genes have been identified and sequenced. Functional analysis of the promoters of the mammalian rRNA genes indicates that despite significant sequence differences, the promoters apparently consist of elements with similar functions. That region of the promoter (--31 to -+6) required for transcription in vitro (5,12,16,26, 43) is referred to as the core promoter element (CPE). Under more stringent conditions, a requirement...

Analysis of the rat ribosomal DNA promoter: characterization of linker-scanning mutants and of the binding of UBF

“…That region of the promoter ( 31 to + 6) sufficient for transcription in vitro, and essential for transcription in vivo is referred to as the core promoter element (CPE). However, when 5'-deletion mutant and wild-type promoters compete against one another under conditions where the levels of the transcription factors are limiting, a requirement for distal sequences becomes apparent (5,12). In vivo experiments demonstrate that constructs with only the CPE are essentially inactive (7).…”

“…Functional analyses of the elements of the promoters of the vertebrate rRNA genes demonstrates that despite significant sequence differences, the promoters appear to consist of at least two or possibly three elements which function homologously (4,5,6). That region of the promoter ( 31 to + 6) sufficient for transcription in vitro, and essential for transcription in vivo is referred to as the core promoter element (CPE).…”

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

“…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). It has been proposed that UBF acts as both an activator, by stabilizing the interaction of the TBP-containing activity and/or RNA polymerase I with the promoter and as an anti-repressor by preventing histone (or other DNA binding factors) mediated repression (35,36).…”

“…In human extracts the pol I transcription activity named SL1 [Selectivity Factor 1; (26)] contains TBP and at least three TBPAssociated Factors (TAFs) (27). In other species the equivalent activity has been named TFID, Factor D, Rib-I or TIF-1B (15,18,(28)(29)(30)(31)(32). 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).…”

“…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.…”