Interaction of SmpB with ribosome from directed hydroxyl radical probing.
To add a tag-peptide for degradation to the nascent polypeptide in a stalled ribosome, an unusual translation called trans-translation is facilitated by transfer-messenger RNA (tmRNA) having an upper half of the tRNA structure and the sequence encoding the tag-peptide except the first alanine. During this event, tmRNA enters the vacant A-site of the stalled ribosome without a codon-anticodon interaction, but with a protein factor SmpB. Here, we studied the sites and modes of binding of SmpB to the ribosome by directed hydroxyl radical probing from Fe(II) tethered to SmpB variants. It revealed two SmpB-binding sites, A-site and P-site, on the ribosome. Each SmpB can be superimposed on the lower half of tRNA behaving in translation. The sites of cleavages from Fe(II) tethered to the C-terminal residues of A-site SmpB are aligned along the mRNA path towards the downstream tunnel, while those of P-site SmpB are found almost exclusively around the region of the codon-anticodon interaction in the P-site. We propose a new model of trans-translation in that the C-terminal tail of SmpB initially recognizes the decoding region and the mRNA path free of mRNA by mimicking mRNA.
Kurita
Daisuke
D
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
Sasaki
Rumi
R
Muto
Akira
A
Himeno
Hyouta
H
D001665:Binding Sites; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D012343:RNA, Transfer; D004492:Edetic Acid