Single-molecule dynamics of the molecular chaperone trigger factor in living cells.
In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to assist the folding of nascent polypeptides. Studies in vitro have provided insights into the function and mechanism of TF. Much is to be elucidated, however, about how TF functions in vivo. Here, we use single-molecule tracking, in combination with genetic manipulations, to study the dynamics and function of TF in living E. coli cells. We find that TF, besides interacting with the 70S ribosome, may also bind to ribosomal subunits and form TF-polypeptide complexes that may include DnaK/DnaJ proteins. The TF-70S ribosome interactions are highly dynamic inside cells, with an average residence time of ∼0.2 s. Our results confirm that the signal recognition particle weakens TF's interaction with the 70S ribosome, and further identify that this weakening mainly results from a change in TF's binding to the 70S ribosome, rather than its unbinding. Moreover, using photoconvertible bimolecular fluorescence complementation, we selectively probe TF2dimers in the cell and show that TF2does not bind to the 70S ribosome but is involved in the post-translational interactions with polypeptides. These findings contribute to the fundamental understanding of molecular chaperones in assisting protein folding in living cells.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Feng
F
Yang
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Tai-Yen
TY
Chen
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Łukasz
Ł
Krzemiński
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Ace George
AG
Santiago
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Won
W
Jung
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, 14853, USA.
Peng
P
Chen
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D012270:Ribosomes / Q000378:metabolism
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010455:Peptides; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase