Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits

Kurt Fredrick, Gary M. Dunny, Harry F. Noller

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Ribosomal protein S7 nucleates folding of the 16 S rRNA 3' major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein Kinase A recognition site and engineered alanine substitutions that target each exposed, conserved residue. We have also deleted conserved features of S7, using its structure to guide our design. By radiolabeling the tag sequence using Protein Kinase A, we are able to track the partitioning of each mutant protein into 30 S, 70 S, and polyribosome fractions in vivo. Overexpression of S7 confers a growth defect, and we observe a striking correlation between this phenotype and proficiency in 30 S subunit assembly among our collection of mutants. We find that the side chain of K35 is required for efficient assembly of S7 into 30 S subunits in vivo, whereas those of at least 17 other conserved exposed residues are not required. In addition, an S7 derivative lacking the N-terminal 17 residues causes ribosomes to accumulate on mRNA to abnormally high levels, indicating that our approach can yield interesting mutant ribosomes. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)379-394
Number of pages16
JournalJournal of Molecular Biology
Volume298
Issue number3
DOIs
StatePublished - May 5 2000

Bibliographical note

Funding Information:
We thank T. Nguyenle for the gift of purified 30 S subunits isolated from MRE600, V. Ares for oligonucleotide synthesis, and M. Costa, G. Culver, J. Diener, J. Helmann, L. Holmberg, A. Kopylov, L. Lancaster, M. Nomura, and K. Wilson for comments on the manuscript. We also thank members of the Dunny laboratory for their generosity toward K.F. during his stay in Minnesota. This work was supported by NIH grant GM17129 and by a grant to the Center for Molecular Biology of RNA from the W. M. Keck Foundation. K.F. was supported by NIH post-doctoral fellowship F32GM19196.

Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.

Keywords

  • 16 S rRNA
  • 30 S subunit
  • Ribosomal protein S7
  • Ribosome assembly
  • str operon

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