Analysis of Tat targeting function and twin-arginine signal peptide activity in Escherichia coli.
The Tat system is a protein export system dedicated to the transport of folded proteins across the prokaryotic cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Proteins are targeted for export by the Tat system via N-terminal signal peptides harbouring an S-R-R-x-F-L-K 'twin-arginine' motif. In this chapter qualitative and quantitative assays for native Tat substrates in the model organism Escherichia coli are described. Genetic screening methods designed to allow the rapid positive selection of Tat signal peptide activity and the first positive selection for mutations that inactivate the Tat pathway are also presented. Finally isothermal titration calorimetry (ITC) methods for measuring the affinity of twin-arginine signal peptide-chaperone interactions are discussed.
Palmer
Tracy
T
Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, Scotland.
Berks
Ben C
BC
Sargent
Frank
F
D002151:Calorimetry; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006864:Hydrogenase / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008957:Models, Genetic; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D011485:Protein Binding
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C408585:twin-arginine translocase complex, E coli; D006864:Hydrogenase; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase