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Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions.
J Mol Biol. 1996 Aug 23; 261(3):357-71.JM

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+Show Affiliations

Howard Hughes Medical Institute, University of Utah Salt Lake City 84112, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8780779

Citation

Adamski, F M., et al. "Ribosomal Protein L9 Interactions With 23 S rRNA: the Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions." Journal of Molecular Biology, vol. 261, no. 3, 1996, pp. 357-71.
Adamski FM, Atkins JF, Gesteland RF. Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions. J Mol Biol. 1996;261(3):357-71.
Adamski, F. M., Atkins, J. F., & Gesteland, R. F. (1996). Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions. Journal of Molecular Biology, 261(3), 357-71.
Adamski FM, Atkins JF, Gesteland RF. Ribosomal Protein L9 Interactions With 23 S rRNA: the Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions. J Mol Biol. 1996 Aug 23;261(3):357-71. PubMed PMID: 8780779.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions. AU - Adamski,F M, AU - Atkins,J F, AU - Gesteland,R F, PY - 1996/8/23/pubmed PY - 1996/8/23/medline PY - 1996/8/23/entrez SP - 357 EP - 71 JF - Journal of molecular biology JO - J Mol Biol VL - 261 IS - 3 N2 - 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. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/8780779/Ribosomal_protein_L9_interactions_with_23_S_rRNA:_the_use_of_a_translational_bypass_assay_to_study_the_effect_of_amino_acid_substitutions_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(96)90469-6 DB - PRIME DP - Unbound Medicine ER -