Ligand binding by Notch receptors triggers a series of proteolytic cleavages 
that liberate the intracellular portion of Notch (ICN) from the cell membrane, 
permitting it to translocate to the nucleus. Nuclear ICN binds to a highly 
conserved DNA-binding transcription factor called CSL (also known as RBP-Jkappa, 
CBF1, Suppressor of Hairless, and Lag-1) and recruits Mastermind-like 
transcriptional co-activators to form a transcriptional activation complex. 
Using bioinformatics tools, we identified a Rel homology region (RHR) within CSL 
that was used as a guide to determine the minimal protein requirements for 
ternary complex formation. The RHR of CSL contains both the N- and C-terminal 
beta-sheet domains (RHR-n and RHR-c) of typical Rel transcription factors, as 
judged by circular dichroism spectra. Binding of monomeric CSL to DNA requires 
the entire RHR of CSL and an additional 125-residue N-terminal sequence, whereas 
binding to ICN requires only the RHR-n domain. Although the RAM (RBP-Jkappa 
(recombination-signal-sequence-binding protein for Jkappa genes)-associated 
molecule) domain of ICN is flexible and relatively unstructured as an isolated 
polypeptide in solution, it associates stably with CSL on DNA. Recruitment of 
Mastermind-like 1 (MAML1) to CSL.ICN complexes on DNA requires inclusion of the 
ankyrin repeat domain of ICN, and N- and C-terminal sequences of CSL extending 
beyond the DNA-binding region. The requirement for cooperative assembly of the 
MAML1.ICN.CSL.DNA complex suggests that a primary function of ICN is to render 
CSL competent for MAML loading. On the basis of our results, we present a 
working structural model for the organization of the MAML1.ICN.CSL.DNA complex.