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Journal of Bacteriology, June 2004, p. 3777-3784, Vol. 186, No. 12
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.12.3777-3784.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Functional Dissection of Escherichia coli Trigger Factor: Unraveling the Function of Individual Domains

G. Kramer,{dagger} A. Rutkowska, R. D. Wegrzyn, H. Patzelt, T. A. Kurz, F. Merz, T. Rauch, S. Vorderwülbecke,{ddagger} E. Deuerling,* and B. Bukau*

Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, D-69120 Heidelberg, Germany

Received 14 January 2004/ Accepted 9 March 2004

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of {Delta}tig {Delta}dnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, {Delta}tig{Delta}dnaK cells expressing the N domain alone grew more slowly and showed less viability than {Delta}tig{Delta}dnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured D-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.

* Corresponding author. Mailing address: Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. Phone: 49-6221-546870. Fax: 49-6221-545894. E-mail for B. Bukau: bukau{at}zmbh.uni-heidelberg.de. E-mail for E. Deuerling: e.deuerling{at}zmbh.uni-heidelberg.de.

{dagger} Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

{ddagger} Present address: Ciphergen Biosystems GmbH, 37085 Goettingen, Germany.

Journal of Bacteriology, June 2004, p. 3777-3784, Vol. 186, No. 12
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.12.3777-3784.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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