Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA.
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.
Ostergaard
P
P
RNA Regulation Centre Institute of Molecular Biology, Copenhagen University, Solvgade 83H, Copenhagen K, DK-1307, Denmark.
Phan
H
H
Johansen
L B
LB
Egebjerg
J
J
Ostergaard
L
L
Porse
B T
BT
Garrett
R A
RA
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli; D007477:Ions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013696:Temperature
D007477:Ions; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; C036298:ribosomal protein L24; C056368:ribosomal protein L4; C049650:ribosomal protein L9