Studies of the complex of ribosomal protein L1 with its binding site in 23S rRNA: Modification -interference, mutagenesis and crosslinking
Interaction of ribosomal protein L1 and the 23S rRNA plays an important role in both the structure and biological activity of the Escherichia coli ribosome. We have minimized the binding site for protein L1 on the 23S rRNA to a 32-nucleotide fragment consisting of helix 77, helix 78, and the conserved sequences that interconnect them. Filter-binding, modification-interference and manganese rescue experiments were used do demonstrate (1) that the absence of a 2′-OH group at position 2122 can disrupt protein L1-23S rRNA interaction, but only if certain other deoxyribonucleotides are present in the transcript and (2) that the Rp phosphoryl oxygens preceding U2122 and A2176 in the rRNA molecule play a role in Ll-23S rRNA interaction through magnesium ion coordination, possibly by participating in magnesium bridges with the protein. The crystal structure of protein L1 from Thermus thermophilus, which closely resembles Escherichia coli L1, consists of two domains that are divided by a deep cleft. It has been suggested that the binding site for RNA is located within this interdomain cleft. Using site-directed mutagenesis it was possible to identify a cluster of conserved amino acids that are crucial for protein-RNA interactions (F37, D42, T216, G218), as well as several amino acids that help to stabilize the complex (K36, P137, N138, K140, H171, K176, M217). All of the mutagenized amino acids are located on the surface of the interdomain cleft. To orient L1 relative to the RNA, crosslinking studies were performed using photoreactive moieties incorporated into the L1 binding site and protein L1 itself. A 4-thiouridine residue at position 2172 and 2-azidoadenosine ligated to the 3′-end of the minimized binding site were shown to form a “zero length” crosslink with protein L1. Whereas the azidophenacyl group attached to position 40 of the protein was demonstrated to map a portion of loop 76/77 on 23S rRNA.