Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA.
In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem-loop in tmRNA during later steps of trans-translation.
Department of Cell and Molecular Biology, BMC, Uppsala University Box 596, S-75 124 Uppsala, Sweden.
Natalia
N
Ivanova
Michael Y
MY
Pavlov
Elli
E
Bouakaz
Måns
M
Ehrenberg
Lovisa Holmberg
LH
Schiavone
D001483:Base Sequence; D001665:Binding Sites; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D023302:Protein Footprinting; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*
D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; C430328:small protein B