title: Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions.
abstract: During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass 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 mutation in the C-terminal domain of Escherichia coli large ribosomal subunit 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 domains that both contain a predicted RNA binding site. We have determined 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 retained 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 nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint 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 gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins 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, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.
authors.affiliation: Howard Hughes Medical Institute, University of Utah Salt Lake City 84112, USA.
authors.forename: F M
authors.initials: FM
authors.lastname: Adamski
authors.forename: J F
authors.initials: JF
authors.lastname: Atkins
authors.forename: R F
authors.initials: RF
authors.lastname: Gesteland
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA
chemical_list: D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins; C049650:ribosomal protein L9