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

Abstract

To investigate the mechanism of transcription of the rat ribosomal DNA (rDNA) promoter, a series of 23 linker-scanning mutants were constructed and assayed in transfected CHO cells and with cell-free extracts. With minor variation, the results of the in vitro and in vivo assays paralleled one another. For example, these assays demonstrated that the mutagenesis of bases from -133 to -124, and those from -106 to -101 of the rDNA promoter significantly inhibited transcription both in vivo and in vitro. Both of these sites lie within the upstream promoter element (UPE) of the rDNA promoter. Several constructs, in particular one that mutated the bases between -49 and -45, were better promoters in vivo than the wild-type promoter. DNAse footprinting experiments with purified UBF, an RNA polymerase I transcription factor, demonstrated the importance of the bases between -106 and -101 for the binding of that factor, providing a positive correlation between the transcription experiments and the binding of UBF to the rDNA promoter.

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

“…2A, lane 3). These findings confirm previous studies that have demonstrated that the guanine at -7 is essential for transcription by RNA polymerase I from the 45S promoter in vivo (34). They also indicate that the expression of pSMECAT must to be due to transcription by RNA polymerase I rather than spurious initiation from cryptic polymerase II or III promoters.…”

Nucleolus: the fascinating nuclear body

“…Each rDNA unit consists of a transcribed sequence and an external non-transcribed spacer (Hadjiolov 1985 ; Liau and Perry 1969 ) in which all the sequences necessary for proper RNA pol I transcription such as proximal promoters, spacer promoters and terminators are located (Hadjiolov 1985 ). In the rDNA promoter two important elements have been described, a CORE element and an upstream control element (UCE) (Haltiner et al 1986 ; Windle and Sollner-Webb 1986 ; Xie et al 1992 ) that function synergistically to recruit a transcriptionally competent RNA pol I complex. This complex contains in addition to RNA pol I, the upstream binding factor (UBF) (Pikaard et al 1989 ; Voit et al 1992 ), the selectivity factor protein complex SL1 (Learned et al 1985 ) also called TIF-1B in mouse cells (Clos et al 1986 ), consisting of the TATA-binding protein (TBP) and four transcription activating factors [TAF I s110, 63, 48 and 41 (Comai et al 1994 ; Gorski et al 2007 ; Zomerdijk et al 1994 )], the transcription initiation factor TIF-IA, the mouse homolog of Rrn3p (Bodem et al 2000 ; Moorefield et al 2000 ) and the transcription termination factor TTF-1 (Bartsch et al 1988 ).…”

Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1

“…pSMECAT-7 was used in place of pSMECAT in some experiments. pSMECAT-7 contains a G to A substitution at 77 in the rDNA promoter which inhibits transcription by RNA polymerase I (Xie et al, 1992). Thus, pSMECAT-7 is a control for the possibility that CAT expression results from transcription by RNA polymerase II or RNA polymerase III.…”

“…These proteins are generated by alternative splicing of the transcripts of one gene (Hisatake et al, 1991;O'Mahony and Rothblum, 1991;Putnam and Pikaard, 1992;Kuhn et al, 1994;Paule, 1994). UBF is not absolutely required for speci®c initiation on the rDNA promoter in vitro, although its addition to UBFdepleted extracts increases the eciency of transcription in a dose dependent manner (Jantzen et al, 1990;Xie et al, 1992;Smith et al, 1990;1993). In addition, overexpression of UBF1 in either immortal cells or primary cultures of cardiomyocytes is sucient to directly increase transcription of a reporter for rDNA transcription (Hannan et al, 1996b;.…”