Structure probing of tmRNA in distinct stages of trans-translation.
Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helper protein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work we used lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery at different steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reacted with Ala-tmRNA*EF-Tu*GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was probed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we also analyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. We observed some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNA conformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA is in complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.
Ivanova
Natalia
N
Department of Cell and Molecular Biology, Uppsala University, S-75124 Uppsala, Sweden.
Lindell
Magnus
M
Pavlov
Michael
M
Holmberg Schiavone
Lovisa
L
Wagner
E Gerhart H
EG
Ehrenberg
Måns
M
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007854:Lead / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D046911:Macromolecular Substances; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA; D007854:Lead; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu