Two ribosomal DNA-binding factors interact with a cluster of motifs on the 5' external transcribed spacer, upstream from the primary pre-rRNA processing site in a higher plant.
In radish the primary processing site in pre-rRNA has been mapped to a TTTTCGCGC sequence (motif P) in the 5' external transcribed spacer (5' ETS) of the ribosomal DNA (rDNA) [Delcasso-Tremousaygue, D., Grellet, F., Panabières, F., Ananiev, E. & Delseny, M. (1988) Eur. J. Biochem. 172, 767-776]. The processing site is just downstream of four similar motifs named A1, A2, A3 and B. The five motifs constitute cluster A123BP. We have described previously that in radish extracts a nuclear protein, nuclear factor B (NF B) specifically binds to motif B [Echeverría, M., Penon, P. & Delseny, M. (1994) Mol. Gen. Genet. 243, 442-452]. Here, by means of electrophoretic-mobility-shift assays, we describe an rDNA-binding activity, nuclear factor D (NF D), that interacts with the A123BP cluster. Using various rDNA probes and competitors we show that NF D binds specifically to the A123 clustered motifs but not to similar B or P motifs. We used sequence-specific DNA-affinity chromatography to separate NF D from NF B. DNase I footprinting was used to map the binding site of NF D on the A123BP cluster and we compared it with that of NF B on the same probe. The footprint of NF D extends from the A1 motif to the 5' end of the NF B-binding site and includes motifs A2 and A3 on each strand. The footprinting of NF B is restricted to motif B and adjacent nucleotides. Thus the NF D-binding and NF B-binding sites are distinct but overlap. These two factors bind with a high specificity to the A123BP cluster in the radish 5' ETS. The possibility that these factors regulate rDNA transcription elongation at the level of the primary pre-rRNA processing site in crucifers is discussed.
Caparros-Ruiz
D
D
Laboratoire de Physiologie et Biologie Moléculaire des Plantes, UMR CNRS 5545, Université de Perpignan, France.
Lahmy
S
S
Piersanti
S
S
Echeverría
M
M
D001483:Base Sequence; D001937:Brassica / Q000235:genetics*; D002846:Chromatography, Affinity; D018983:DNA Footprinting; D004275:DNA, Ribosomal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013698:Templates, Genetic
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012335:RNA, Ribosomal