Nucleic Acids Research Advance Access originally published online on May 27, 2009
Nucleic Acids Research 2009 37(13):4482-4497; doi:10.1093/nar/gkp419
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Nucleic Acids Research, 2009, Vol. 37, No. 13 4482-4497
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gene Regulation, Chromatin and Epigenetics |
Construction and functional analyses of a comprehensive 
54 site-directed mutant library using alanine–cysteine mutagenesis
1National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China, 2Division of Investigative Sciences, Faculty of Medicine, Flowers Building and 3Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK
*To whom correspondence should be addressed. Tel: +86 10 6275 8490; Fax: +86 10 6275 6325; Email: wangyp{at}pku.edu.cn
Received March 9, 2009. Revised May 5, 2009. Accepted May 6, 2009.
The 
54 factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. 54 is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define 54 residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of 54 was constructed by alanine–cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)54 were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of 54 were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased 54 isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after 54 isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the 54-core RNAP interface changes upon activation.