Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA

J Mol Biol. 1998 Nov 27;284(2):227-40. doi: 10.1006/jmbi.1998.2185.

Abstract

The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one primary binding protein associated with each RNA domain and additional proteins assembled to domains I, II, V and VI. For all the combinations of two to five domains, enhanced assembly yields and/or new proteins were observed primarily to those transcripts containing either domains I+II or domains V+VI. This indicates that there are two major protein assembly centres located at the ends of the 23 S rRNA, which is consistent with an earlier view that in vitro protein assembly nucleates around proteins L24 and L3. Although similar protein assembly patterns were observed over a range of temperature and magnesium concentrations, protein L2 assembled strongly with domains II and IV at 4-8 mM Mg2+ (the first step of the two-step reconstitution procedure) and with domain IV alone at higher Mg2+ concentrations (the second step). It is proposed that this change in protein-RNA binding provides a basis for the two-step reconstitution in vitro. A chemical footprinting approach was employed on the reconstituted protein-domain complexes to localize a putative L4 binding region within domain I to a region that is partially co-structural with the site on the L4-mRNA where L4 binds and inhibits its own translation. A similar approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Escherichia coli
  • Ions
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • RNA, Bacterial / metabolism*
  • RNA, Ribosomal, 23S / metabolism*
  • RNA, Ribosomal, 5S / metabolism*
  • Ribosomal Proteins / metabolism*
  • Ribosomes / metabolism*
  • Temperature

Substances

  • Ions
  • RNA, Bacterial
  • RNA, Ribosomal, 23S
  • RNA, Ribosomal, 5S
  • Ribosomal Proteins
  • ribosomal protein L24
  • ribosomal protein L3
  • ribosomal protein L4
  • ribosomal protein L9