Sequence specificity

Artificial endonucleases, ie, molecules able to cleave double-stranded DNA at a specific sequence, have also been developed. These endonucleases can be obtained by attaching a chemically reactive group to a sequence-specific oligonucleotide. When the oligonucleotide is bound to its complementary sequence, the activation of the reactive group results in double-stranded DNA cleavage. 

Figure 7.8 Sequence-specific recognition sites in the major groove of DNA for three restriction enzymes—Eco RI, Bal I, and Sma I. The DNA sequences that are recognized by these enzymes ate represented by tbe color code defined in Figure 7.7.

Bacteriophage repressor proteins provide excellent examples of sequence-specific interactions between the side chains of a protein and bases lining the floor of the major groove of B-DNA. As we shall see, to fit the protein s recognition module into this groove it has to be made even wider in other words, the B-DNA has to be distorted. 

Seeman, N.C., Rosenberg, J.M., Rich, A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc. Natl. Acad. Sci. USA 73 804-809, 1976. 

Figure 8.8 The DNA-binding heiix-turn-helix motif in lambda Cro. Ca positions of the amino acids in this motif have been projected onto a plane and the two helices outlined. The second helix (red) is called the recognition helix because it is involved in sequence-specific recognition of DNA.

Sequence-specific protein-DNA interactions recognize operator regions... 

Figure 8.15 Sequence-specific protein-DNA interactions provide a general recognition signal for operator regions in 434 bacteriophage, (a) In this complex between 434 repressor fragment and a synthetic DNA there are two glutamine residues (28 and 29) at the beginning of the recognition helix in the helix-turn-helix motif that provide such interactions with the first three base pairs of the operator region.

Steitz, T.A. Stmctural studies of protein-nucleic acid interaction the sources of sequence-specific binding. 

Wharton, R.P., Brown, E.L., Ptashne, M. Substituting an a helix switches the sequence-specific DNA interactions of a repressor. Cell 38 361-369, 1984. 

The promoter proximal elements are usually 100 to 200 base pairs long and relatively close to the site of initiation of transcription. Within each of these elements there are DNA sequences specifically recognized by several different transcription factors which either interact directly with the preinitiation complex or indirectly through other proteins. 

Most sequence-specific regulatory proteins bind to their DNA targets by presenting an a helix or a pair of antiparallel p strands to the major groove of DNA. Recognition of the TATA box by TBP is therefore exceptional it utilizes a concave pleated sheet protein surface that interacts with the minor groove of DNA. Since the minor groove has very few sequence-specific... 

The only sequence-specific hydrogen bonds between TBP side chains and the bases in the minor groove occur at the very center of the TATA box (Figure 9.7). The amide groups of two asparagine side chains donate four hydrogen bonds, two each to adjacent bases on the same DNA strand (Asn 69... 

Figure 9.7 Sequence specific interactions between TBP and the TATA box. Asn 69 and Thr 124 from one domain and the equivalent residues Asn 159 and Thr 215 from the second domain interact with the palindromic TATA sequence of the central region of the TATA box.

Like Thr 124 and Thr 215, the Asn 69 and Asn 159 residues occupy equivalent positions in the two homologous motifs of TBP. By analogy with the symmetric binding of a dimeric repressor molecule to a palindromic sequence described in Chapter 8, the two motifs of TBP form symmetric sequence-specific hydrogen bonds to the quasi-palindromic DNA sequence at the center of the TATA box. The consensus TATA-box sequence has an A-T base pair at position 4, but either a T-A or an A-T base pair at the symmetry-related position 5, and the sequence is, therefore, not strictly palindromic. However, the hydrogen bonds in the minor groove can be formed equally well to an A-T base pair or to a T-A base pair, because 02 of thymine and N3 of adenine occupy nearly stereochemically equivalent positions, and it is sufficient, therefore, for the consensus sequence of the TATA box to be quasi-palindromic. 

TFIIB is arranged in two domains, both of which have the cyclin fold described in Chapter 6. Both domains bind to the TBP-TATA box complex at the C-terminal stirrup and helix of TBP. The phosphate and sugar moities of DNA form extensive non-sequence-specific contacts with TFIIB both upstream and downstream of the middle of the TATA box. 

Figure 9.10 Schematic diagrams illustrating the complex between DNA (orange) and one monomer of the homeodomain. The recognition helix (red) binds in the major groove of DNA and provides the sequence-specific interactions with bases in the DNA. The N-terminus (green) binds in the minor groove on the opposite side of the DNA molecule and arginine side chains make nonspecific interactions with the phosphate groups of the DNA. (Adapted from C.R. Kissinger et al Cell 63 579-590, 1990.)...

One of the most important molecular functions of p53 is therefore to act as an activator of p21 transcription. The wild-type protein binds to specific DNA sequences, whereas tumor-derived p53 mutants are defective in sequence-specific DNA binding and consequently cannot activate the transcription of p5 3-con trolled genes. As we will see more than half of the over one thousand different mutations found in p53 involve amino acids which are directly or indirectly associated with DNA binding. 

The polypeptide chain of p53 is divided in three domains, each with its own function (Figure 9.16). Like many other transcription factors, p53 has an N-terminal activation domain followed by a DNA-binding domain, while the C-terminal 100 residues form an oligomerization domain involved in the formation of the p53 tetramers. Mutants lacking the C-terminal domain do not form tetramers, but the monomeric mutant molecules retain their sequence-specific DNA-binding properties in vitro. 

Figure 9.19 shows the sequence of the DNA that was used for the structure determination of the p53-DNA complex the bases involved in sequence-specific binding to the protein are shaded. One molecule of the DNA-bind-ing domain of p53 binds to the minor and the major grooves of the DNA making sequence-specific interactions with both strands (Figure 9.20). 

Figure 9.19 Nucleotide sequence of the 21-base pair DNA fragment cocrystalUzed with the DNA-binding domain of p53. The p53 binds in a sequence-specific manner to the shaded region.

Figure 9.20 Diagram iliustrating the sequence-specific interactions between DNA and p53. The C-terminai a helix and loop LI of p53 bind in the major groove of the DNA. Arg 280 from the a helix and Lys 120 from LI form important specific interactions with bases of the DNA. In addition, Arg 248 from loop L3 binds to the DNA in the minor groove. (Adapted from Y. Cho et al.. Science 265 346-355, 1994.)...

Interactions that are not sequence specific are also an Important part of the binding and occur between the sugar and phosphate residues of the DNA and the side-chain and main-chain atoms of the protein. In the crystals the DNA fragment retains the B-DNA structure with only minor distortions. 

The 12 residues between the second cysteine zinc ligand and the first histidine ligand of the classic zinc finger motif form the "finger region". Structurally, this region comprises the second p strand, the N-terminal half of the helix and the two residues that form the turn between the p strand and the helix. This is the region of the polypeptide chain that forms the main interaction area with DNA and these interactions are both sequence specific. 

Figure 10.4 Detailed view of the binding of the second zinc finger of Zif 268 to DNA. Two side chains, Arg 46 and His 49, form sequence-specific interactions with DNA. There are also three nonspecific interactions between phosphate groups of the DNA and the side chains of Arg 42, Ser 45, and His 53.

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