Our research efforts have focused on elucidation of the molecular nature of interactions between the RNA and the protein in the 5S rRNA-Ll protein (RNP) complex, an early ribosome assembly intermediate. Previous scanning mutagenesis studies of the protein have identified multiple amino acids that are important for RNA binding and assembly into ribosomes. For example, replacement of both residues K270 and K271 in protein LI by arginine or aspartic acid residues leads to cell lethality. Here we describe studies using a genetic approach to identify interacting portions of the intact protein LI. An intragenic suppressor revenant which contains an E257K substitution (in addition to the primary mutations) was identified for the K270,271R mutant. An E257R revenant for the K270.271R mutant was also identified. Both revenant proteins bound RNA in vitro albeit with a reduced affinity (50-60% of the wild-type) and were assembled into the 60S ribosomal subunits in vivo. Additional substitutions of E257 were produced by site-directed mutagenesis. The ability of each second-site mutation to suppress the lethal effect of the double K270.271R (or K270.271E) mutation in vivo was also examined E257G, V, and W substitutions could suppress the K270.271R mutation and E257A, G, and V substitutions could suppress the K270.271E mutation. The present results provide experimental evidence for an interaction between two distal segments of protein LI.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology