title: Target-directed proteolysis at the ribosome.
abstract: Target directed proteolysis allows specific processing of proteins in vivo. This method uses tobacco etch virus (TEV) NIa protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed.
authors.affiliation: School of Biosciences, Cardiff University, Cardiff CF10 3US, UK.
authors.forename: Tanja
authors.initials: T
authors.lastname: Henrichs
authors.forename: Natasha
authors.initials: N
authors.lastname: Mikhaleva
authors.forename: Charlotte
authors.initials: C
authors.lastname: Conz
authors.forename: Elke
authors.initials: E
authors.lastname: Deuerling
authors.forename: Dana
authors.initials: D
authors.lastname: Boyd
authors.forename: Adrian
authors.initials: A
authors.lastname: Zelazny
authors.forename: Eitan
authors.initials: E
authors.lastname: Bibi
authors.forename: Nenad
authors.initials: N
authors.lastname: Ban
authors.forename: Michael
authors.initials: M
authors.lastname: Ehrmann
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D018271:Signal Recognition Particle / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; C087406:FtsY protein, Bacteria; D026901:Membrane Transport Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; D010450:Endopeptidases; C099488:TEV protease; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins