Assembly of proteins and 5S rRNA to transcripts of the major structural domains of 23S rRNA
Research output: Contribution to journal › Journal article › Research › peer-review
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.
Original language | English |
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Journal | Journal of Molecular Biology |
Volume | 284 |
Issue number | 2 |
Pages (from-to) | 227-240 |
Number of pages | 13 |
ISSN | 0022-2836 |
DOIs | |
Publication status | Published - 1 Nov 1998 |
Bibliographical note
Keywords: Base Sequence; Binding Sites; Escherichia coli; Ions; Models, Molecular; Molecular Sequence Data; Protein Binding; RNA, Bacterial; RNA, Ribosomal, 23S; RNA, Ribosomal, 5S; Ribosomal Proteins; Ribosomes; Temperature
ID: 217315