Capítulo I. Fundamentos teóricos-metodológicos que sustenta la Educación Ambiental
Capítulo 2. Sistema de actividades educativas desde la concepción comunitaria para potenciar la
2.1. El Autodesarrollo Comunitario: metodología que sustenta la propuesta
The interaction of SmtB with DNA has been the subject of a number of studies over the last two decades (Morby et al. 1993; Erbe et al. 1995; Turner et al. 1996; Kar et al. 2001; VanZile et al. 2002a). The DNA recognition helices within SmtB have been identified, along with two separate SmtB binding sites within the operator-promoter sequence, (refer to Chapter One for details). No high resolution structure has been obtained, either by NMR or X-ray crystallography of any SmtB: DNA complex. The mechanism of SmtB binding has therefore not been fully characterised, although a number of models have been proposed.
Elucidation of the first crystal structure of apo-SmtB (Cook et al. 1998), combined with the use of methylation interference assays and bioinformatics, led to a greater understanding of possible interactions between SmtB2 and DNA. Cook et al.
superimposed the predicted binding helices of SmtB upon the known DNA binding helices of different H-T-H proteins. The use of hepatocyte nuclear factor 3 (HNF-3), as a template, presented the most convincing model in which both ends of SmtB bound within two consecutive major grooves of the DNA. This binding event was predicted to impose a 30 ° bend in the DNA and to involve the SmtB residues Cys 61 and His 97 (Cook et al. 1998).
A combination of sedimentation velocity data, interference assays (Kar et al. 2001) and fluorescence anisotropy (VanZile et al. 2002a) have since updated the initial model. The current model postulates that both the S2/S1 and S4/S3 sites have the ability to bind up to two SmtB dimers. It remains unclear, however, whether a SmtB tetramer is formed on the DNA or whether two SmtB dimers bind on either side of the DNA, Figure 4.1A. Kar et al. based their sequential binding model upon the formation of a tetramer at the S2/S1 site. This tetramer forms a bridge to the S3 site, which is only displaced after another SmtB dimer binds to the S4 site (Kar et al. 2001). Although apo-SmtB is known to form weak tetramers in solution (Kar et al. 1997), the experimental binding affinities of SmtB dimers, determined by fluorescence anisotropy, were found to increase with DNA length. This evidence
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SmtB
C A C AT G A AC A G T T A T T C A G A T A G T G T A C T T G T C A A T A A G T C T A TSmtB
A
SmtB
C A C AT G A AC A G T T A T T C A G A T A G T G T A C T T G T C A A T A A G T C T A TSmtB
B
supported the proposed complex architecture of SmtB dimers binding on either side of DNA, Figure 4.1B, since the addition of base pairs would increase the interaction surface (VanZile et al. 2002a).
Figure 4.1: Schematic displaying the two possible SmtB: DNA complexes
A SmtB tetramer bound to the DNA (A) or two SmtB dimers on either side of the DNA (B). The 5'- TGAA motifs are shaded in red.
In this model the SmtB dimers were proposed to be centred on a 5'- TGAA motif, Figure 4.1B, in contrast with each end interacting with the sequence, Figure 4.1A. This off-centred binding has also been documented for other ArsR/SmtB members. One dimer of CadC was found to bind to its operator-promotor sequence in this fashion, centred over a 5'-TCAA motif (Endo and Silver 1995). A further dimer has more recently been discovered to bind under low NaCl conditions (Busenlehner et al. 2002). The sensing and DNA binding of CadC was found to be different to that of SmtB. Since CadC is an α3N sensor (Busenlehner et al. 2002) instead of an α5 sensor, its DNA binding 12-2-12 repeat would have a different core motif (Busenlehner et al. 2003). This implies that a different mode of DNA binding may be involved.
CzrA, a homologous protein to SmtB, has more recently been studied in a complex with a 28 bp duplex DNA using NMR (Arunkumar et al. 2009). Unlike CadC, CzrA is an α5 sensor which senses zinc ions. This allowed a more accurate comparison to be made between the CzrA:DNA and SmtB:DNA complexes. Like SmtB, two CzrA dimers (note CzrA = ZntR), were found to have the ability to bind to a single 12-2- 12 inverted repeat at a high affinity (Busenlehner et al. 2003; Lee et al. 2006).
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..T G A AC A A A T A T T C A..
..A C T T G T T T A T A A G T..
CzrA
Within the NMR CzrA:DNA complex, only a 1:1 CzrA dimer:DNA stoichiometry could be achieved and the bound DNA structure remained unknown. The model produced, suggested that the interaction of the αR helices with the 5'- TGAA motifs, Figure 4.2, bent the DNA by 35 ° (Arunkumar et al. 2009). This bend in the DNA corresponded well to the original SmtB:DNA binding model proposed by Cook et al. (Cook et al. 1998).
Figure 4.2: Schematic displaying the CzrA: DNA complex
The 5'- TGAA motifs, thought to interact with the αR helix, are shaded in red.
Limited information regarding the CzrA:DNA interface was obtained, and due to sequence conservation, was thought to be applicable to other α5 sensors, such as SmtB. This work revealed the involvement of CzrA residues, with bracketed SmtB equivalents; Q53 (E73), V42 (V62), H58 (H78), S54 (S74) and S57 (S77) (Arunkumar et al. 2009). These residues lie predominantly within the αR helix although V62 revealed a possible contact with the α3 helix.