A yeast ribosomal DNA-binding protein that binds to the rDNA enhancer and also close to the site of Pol I transcription initiation is not important for enhancer functioning.
Using the gel retardation assay we have identified a protein that can specifically bind to a site within the enhancer of the 37S pre-ribosomal RNA operon in yeast, as well as to a site 210 bp upstream of the site of transcription initiation of this operon. This protein (RBP1) has been partially purified by means of heparin-agarose chromatography and protects 20 bp in the rDNA enhancer, and 25 bp in the initiation region, against DNase I in an in vitro footprinting assay. In vivo footprinting studies using methylation of intact yeast cells with dimethylsulphate, indicate that the same binding sites are occupied in vivo as well. Deletions that abolish binding of RBP1 to the enhancer in vitro, as well as linker insertions into the RBP1 binding site in the initiation region that strongly diminish in vitro binding of RBP1, have no effect whatsoever on the enhancement of rDNA transcription in vivo. This was studied by deletion/mutation of the RBP1 binding site in vitro in an artificial ribosomal minigene and measuring the effect on the minigene transcription in vivo in yeast cells, transformed with the deleted/mutated minigenes. It can therefore be concluded that binding of RBP1 is not an important parameter in the functioning of the rDNA enhancer in yeast. Using the same minigene system we also show that RBP1 is not involved in termination of RNA polymerase I (Pol I) transcription at the main terminator T2.
Kulkens
T
T
Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands.
van Heerikhuizen
H
H
Klootwijk
J
J
Oliemans
J
J
Planta
R J
RJ
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002845:Chromatography; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004275:DNA, Ribosomal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D012318:RNA Polymerase I / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D012318:RNA Polymerase I