Premium
The tmRNA system for ribosome rescue and targeted protein degradation.
Author(s) -
Sauer Robert T.,
McGinness Kathleen,
Martin Andreas,
Farrell Chris,
Moore Sean,
Baker Tania A.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1474
Subject(s) - aaa proteins , ribosome , atp hydrolysis , signal transducing adaptor protein , proteases , biochemistry , proteolysis , microbiology and biotechnology , protein degradation , atpase , protease , chemistry , biophysics , biology , enzyme , rna , phosphorylation , gene
Stalled bacterial ribosomes are recognized and rescued by the tmRNA/SmpB system, resulting in addition of an 11‐residue ssrA tag at the C‐terminus of the protein being synthesized. This ssrA‐tagged protein is subsequently degraded by energy‐dependent cytoplasmic proteases. A specialized adaptor protein (SspB) binds ssrA‐tagged proteins and delivers them to the ClpXP protease for degradation. ClpX is an ATP‐fueled hexameric machine that binds to the ssrA tag, denatures the attached protein if necessary, and then translocates the denatured polypeptide through a central pore and into the degradation chamber of ClpP, a barrel‐shaped peptidase for proteolysis. Studies of the mechanisms of tag‐mediated and adaptor‐mediated substrate recognition allowed us to design a modified ssrA tag and to engineer a system that permits controlled SspB‐dependent protein degradation of any suitably tagged bacterial protein. ClpX is a member of the AAA+ ATPase family. As a group, the enzymes use ATP hydrolysis to power the mechanical denaturation, disassembly, and transport of macromolecules. We designed single‐chain “hexamers” of ClpX, allowing the functional roles of individual subunits within the ring to be dissected. These studies rule out concerted and strict sequential models of ATP hydrolysis and suggest that a probabilistic mechanism is used to translocate denatured polypeptides despite radically different sequences and physical properties. These studies have important implications for protein‐quality control and for the mechanisms of AAA+ molecular machines.