Structural determinants in 5S RNA and TFIIIA for 7S RNP formation

C2H2-type zinc-finger modules define a unique structural motif, which is ca pable of forming specific complexes with both DNA and RNA. While the princi ples governing DNA binding have been defined in great detail, the mode of R NA recognition remains only poorly understood. In the absence of informatio n from three-dimensional structural analysis of a zinc-finger/RNA complex, we have performed it number of biochemical studies to gain further insight into the molecular details of the interaction of 5S ribosomal RNA with the zinc-finger protein TFIIIA. Previous work had indicated that zinc finger 6 of TFIIIA contacts 5S RNA in close proximity or directly in the loop-A regi on (nucleotides 10-13). Permutation analysis of this sequence reveals that three of the four nucleotides are of vital importance for RNA recognition. Exchange of unusual and therefore characteristic aromatic residues in finge r 6 against aliphatic or other aromatic amino acids reveals that the aromat ic character of tryptophan 177 is essential for RNA recognition. Associatio n with helix V in 5S RNA appears to involve specific contacts with the phos phate backbone, as evidenced by ethylation-interference assays. Introductio n of multiple internal and 3'-terminal as well as 5'-terminal deletions acc ompanied by stabilizing sequence substitutions defines a minimal RNA fragme nt that is sufficient fur TFIIIA binding. This RNA molecule includes a trun cated/ mutated helix I, helix II and helix V, as well as structurally intac t loops A and E. permutation analysis of the loop-E region emphasizes its i mportance for TFIIIA recognition.