Nucleic Acids Research, Vol 25, Issue 11 2047-2054, Copyright © 1997 by Oxford University Press
J Chen, S Pongor and A Simoncsits
Single-chain derivatives of the phage 434 repressor, termed single- chain
repressors, contain covalently dimerized DNA-binding domains (DBD) which
are connected with a peptide linker in a head-to-tail arrangement. The
prototype RR69 contains two wild-type DBDs, while RR*69 contains a
wild-type and an engineered DBD. In this latter domain, the DNA- contacting
amino acids of thealpha3 helix of the 434 repressor are replaced by the
corresponding residues of the related P22 repressor. We have used binding
site selection, targeted mutagenesis and binding affinity studies to define
the optimum DNA recognition sequence for these single-chain proteins. It is
shown that RR69 recognizes DNA sequences containing the consensus boxes of
the 434 operators in a palindromic arrangement, and that RR*69 optimally
binds to non-palindromic sequences containing a 434 operator box and a TTAA
box of which the latter is present in most P22 operators. The spacing of
these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both
single-chain repressors, similar to that of the 434 repressor, is
influenced indirectly by the sequence of the non-contacted, spacer region.
Thus, high affinity binding is dependent on both direct and indirect
recognition. Nonetheless, the single-chain framework can accommodate
certain substitutions to obtain altered DNA-binding specificity and RR*69
represents an example for the combination of altered direct and unchanged
indirect readout mechanisms.
ARTICLES
Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non- contacted base pairs
International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano 99, I-34012 Trieste, Italy.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Guarnaccia, B. Raman, S. Zahariev, A. Simoncsits, and S. Pongor DNA-mediated assembly of weakly interacting DNA-binding protein subunits: in vitro recruitment of phage 434 repressor and yeast GCN4 DNA-binding domains Nucleic Acids Res., September 23, 2004; 32(17): 4992 - 5002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. Pawlowski and G. B. Koudelka The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor Is the DNA-Bound Dimer J. Bacteriol., January 1, 2004; 186(1): 1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. P. Papp, T. Nagy, S. Ferenczi, P. Elo, Z. Csiszovszki, Z. Buzas, A. Patthy, and L. Orosz Binding sites of different geometries for the 16-3 phage repressor PNAS, June 25, 2002; 99(13): 8790 - 8795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Liang, J. Chen, M.-L. Tjornhammar, S. Pongor, and A. Simoncsits Modular construction of extended DNA recognition surfaces: mutant DNA-binding domains of the 434 repressor as building blocks Protein Eng. Des. Sel., August 1, 2001; 14(8): 591 - 599. [Abstract] [Full Text] [PDF]
|
|