Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

Abstract

A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three-to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20-to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.The cellular machinery responsible for RNA synthesis is designed to respond to a variety of specific signals so that genes can be transcribed and expressed in a controlled fashion. For example, the synthesis of rRNA is sensitive to a variety of physiological signals such as response to nutrient starvation, regulation by cell cycle, the state of cell proliferation, and invasion by viral infection (6,9,17,24,26,27,31). In addition, an interesting and unexpected property of rRNA transcription is the inability of the transcriptional machinery of one species to initiate transcription from the promoter of divergent species. Thus, whereas both sets of rRNA genes can be transcribed in an interspecific rodent cell hybrid, the genes from only one species are expressed in rodent-human cell hybrids (22,32). This species specificity is mimicked in vitro in that extracts from one species are capable of transcribing only those rDNA templates that are derived from the same or closely related species (5,12,14). The limited homology in the DNA sequence surrounding the transcription start site of mammalian rRNA genes suggests that species-specific promoter recognition is likely to involve distinctive rRNA promoter elements.Studies with fractionated enzyme components derived from mammalian cell extracts have allowed the identification of at least two distinct activities that are necesary to reconstitute accurate and efficient initiation of transcription from the human ribosomal...

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

“…Haltiner et al (4) reached a similar conclusion from their study of the effects of linker-scanning mutagenesis of the human rDNA promoter on promoter activity in vitro, i.e. they found a relatively neutral region that stretched from -107 to -45, that was flanked by domains more sensitive to mutagenesis.…”

“…The sequence written is that of the (+) strand, the complement of the sequence deterrnined. Figure 1 and the pseudo wild-type promoter (4 Figure 6) using UBF purified through the CM-Sephadex column chromatography step (5). BSM -106/-101 significantly reduced the binding of UBF to the rat rDNA promoter on both the upper and lower strands ( Figure 6, panels A and B).…”

“…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 studies on the human promoter clearly indicated several domains important for transcription. However, several of the linker-scanning mutants examined included distancealtering mutations (4,27 …”

“…Two vertebrate rDNA promoters have been subjected to linkerscanning analysis, the human and the X. laevis promoters (4,6,27,28). The studies on the human promoter clearly indicated several domains important for transcription.…”