1. Volume 22, Issue 1, Pages 73-81 (April 2006)
The Structure of Phage ϕ29 Transcription Regulator p4-DNA Complex Reveals an N- Hook Motif for DNA Binding 
Daniel Badia, Ana Camacho, Laura Pérez-Lago, Cristina Escandón, Margarita Salas, Miquel Coll 
Molecular Cell 
Volume 22, Issue 1, Pages (April 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Detail of the ϕ29 Genome Intergenic Region between the Early Promoters A2c and A2b and the Late Promoter A3
In this intergenic region, there are four p4 binding sites. Sites 1 and 3 overlay with −35 promoter boxes of the early promoters. Two guanine residues essential for specific recognition are boxed in blue at each site except at site 4, where one of the Gs is lacking. The −10 and −35 elements of the promoters are in boxes. Protein p4 dimers are drawn as elliptical objects with their two apical N-hook recognition motifs. Red lines indicate protected nucleotides from hydroxyl radical attack after p4 binding. The boxed area encompassing site 3 and part of site 4 indicates the DNA sequence used for the p4-DNA complex crystal.
Molecular Cell  , 73-81DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2 Structure of the p4 Monomer and the N-Hook
(A) Overall structure of the protein p4 monomer in a ribbon representation. Helix α4 is only seen in the DNA bound structure.
(B) Stick representation of the N-hook DNA binding motif showing important residues for its folding or DNA binding. H bonds are indicated with dashed lines.
(C) Two views, 90° rotated, of the p4 dimer electrostatic potential surface. The N-hook DNA recognition motifs are clearly seen sticking out at the tips of the dimer. Three positively charged patches, where interactions with the DNA occur, are apparent along the S-shaped dimer (right).
Molecular Cell  , 73-81DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3 Structure of the p4 Dimer-DNA Complex
(A) Representation of the p4 dimer (ribbons) in complex with a 41-mer DNA oligonucleotide (sticks) encompassing the whole site 3 sequence and part of site 4 (not covered by the protein).
(B) Schematic representation of the 41-mer DNA with structural characteristics of the double helix indicated for the region comprising site 3, where the protein dimmer interacts. The 2-fold axis of the inverted repeat is shown as a black ellipsoid. The orientation of the DNA is the same as in (A).
Molecular Cell  , 73-81DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4 p4-DNA Interactions
(A) Detail of the DNA recognition by the N-hook and other nearby interactions of the N terminus of α1.
(B) Detail of the protein-DNA interactions at the dimerization region, involving positively charged residues of the C terminus of helices α1 of both p4 monomers.
(C) Scheme of half site 3 showing the protein-DNA interactions. H bond interactions are shown as dashed lines. Sequence-specific contacts are highlighted with a star at the DNA-recognized atom. A van der Waals interaction is indicated by a continuous line. The 2-fold axis of the inverted repeat is shown as a black ellipsoid.
Molecular Cell  , 73-81DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Band-Shift Assays
The protein p4-site 3 complex has slower mobility than free DNA when run in polyacrylamide gels. This figure shows that mutants K3A, Q5A, K51A, and R54A form p4/DNA complex at similar or higher (Q5A) extent, as does the wild-type protein. In contrast, 6-fold more protein was required for complex formation with mutant H10A, and a 13-fold higher amount of protein was needed when mutants K36A and K76A were used. Mutants T4A, R6A, and Y33A had severely reduced its ability to interact with site 3. Protein p4 concentration is given as dimers.
Molecular Cell  , 73-81DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions