RIBOSOMAL-PROTEIN L9 INTERACTIONS WITH 23-S RIBOSOMAL-RNA - THE USE OF A TRANSLATIONAL BYPASS ASSAY TO STUDY THE EFFECT OF AMINO-ACID SUBSTITUTIONS

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypas s is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutati on in the C-terminal domain of Escherichia coli large ribosomal subuni t protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal dom ains that both contain a predicted RNA binding site. We have determine d two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is r etained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucl eotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprin t assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gen e 60 mRNA has been used as an assay to probe the importance of particu lar L9 amino acids for function. Amino acid substitutions in the C-ter minal domain are shown to partially restore bypass. These mutant L9 pr oteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, sub stitutions of amino acids in the N-terminal domain, which greatly redu ce RNA binding, do not restore bypass. The latter mutants have complet ely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this m utant is similar to that observed for the C-terminal domain mutants th at partially restore bypass. (C) 1996 Academic Press Limited.