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, R, Panabieres, R, 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 [Echeverria, 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.