Principles of Gene Regulation:- Regulatory Proteins Have Discrete DNA-Binding Domains
Regulatory proteins generally bind to specific DNA sequences. Their affinity for these target sequences is roughly 104 to 106 times higher than their affinity for any other DNA sequences. Most regulatory proteins have discrete DNA-binding domains containing sub structures that interact closely and specifically with the DNA. These binding domains usually include one or more of a relatively small group of recognizable and characteristic structural motifs. To bind specifically to DNA sequences, regulatory proteins must recognize surface features on the DNA. Most of the chemical groups that differ among the four bases and thus permit discrimination between base pairs are hydrogen-bond donor and acceptor groups exposed in the major groove of DNA (Fig. 1), and most of the protein-DNA contacts that impart specificity are hydro gen bonds. A notable exception is the nonpolar surface near C-5 of pyrimidines, where thymine is readily distinguished from cytosine by its protruding methyl group. Protein-DNA contacts are also possible in the minor groove of the DNA, but the hydrogen-bonding patterns here generally do not allow ready discrimination between base pairs. Within regulatory proteins, the amino acid side chains most often hydrogen-bonding to bases in the DNA are those of Asn, Gln, Glu, Lys, and Arg residues. Is there a simple recognition code in which a particular amino acid always pairs with a particular base? The two hydrogen bonds that can form between Gln or Asn and the N6 and N-7 positions of adenine cannot form with any other base. And an Arg residue can form two hydrogen bonds with N-7 and O6 of guanine (Fig. 2). Examination of the structures of many DNA-binding proteins, however, has shown that a protein can recognize each base pair in more than one way, leading to the conclusion that there is no simple amino acid–base code. For some proteins, the Gln-adenine interaction can specify A=T base pairs, but in others a van der Waals pocket for the methyl group of thymine can recognize A=T base pairs. Researchers cannot yet examine the structure of a DNA-binding protein and infer the DNA sequence to which it binds.
To interact with bases in the major groove of DNA, a protein requires a relatively small structure that can stably protrude from the protein surface. The DNA binding domains of regulatory proteins tend to be small (60 to 90 amino acid residues), and the structural motifs within these domains that are actually in contact with the DNA are smaller still. Many small proteins are unstable because of their limited capacity to form layers of structure to bury hydrophobic groups . The DNA-binding motifs provide either a very compact stable structure or a way of allowing a segment of protein to protrude from the protein surface.
FIGURE 1 Groups in DNA available for protein binding. Shown here are functional groups on all four base pairs that are displayed in the major and minor grooves of DNA. Groups that can be used for base-pair recognition by proteins are shown in red.
FIGURE 2 Two examples of specific amino acid–base pair inter actions that have been observed in DNA-protein binding.
The DNA-binding sites for regulatory proteins are often inverted repeats of a short DNA sequence (a palindrome) at which multiple (usually two) subunits of a regulatory protein bind cooperatively. The Lac repressor is unusual in that it functions as a tetramer, with two dimers tethered together at the end distant from the DNA-binding sites . An E. coli cell normally contains about 20 tetramers of the Lac repressor. Each of the tethered dimers separately binds to a palindromic operator sequence, in contact with 17 bp of a 22 bp region in the lac operon (Fig.3). And each of the tethered dimers can independently bind to an operator sequence, with one generally binding to O1 and the other to O2 or O3 . The symmetry of the O1 operator sequence corresponds to the twofold axis of symmetry of two paired Lac repressor subunits. The tetrameric Lac repressor binds to its operator sequences in vivo with an estimated dissociation constant of about 10-10 M. The repressor discriminates between the operators and other sequences by a factor of about 106, so binding to these few base pairs among the 4.6 million or so of the E. coli chromosome is highly specific.
Several DNA-binding motifs have been described, but here we focus on two that play prominent roles in the binding of DNA by regulatory proteins: the helix turn-helix and the zinc finger. We also consider a type of DNA-binding domain—the homeodomain—found in some eukaryotic proteins.
FIGURE 3 Relationship between the lac operator sequence O1 and the lac promoter. The bases shaded beige exhibit twofold (palindromic) symmetry about the axis indicated by the dashed vertical line.
Helix-Turn-Helix This DNA-binding motif is crucial to the interaction of many prokaryotic regulatory proteins with DNA, and similar motifs occur in some eukaryotic regulatory proteins. The helix-turn-helix motif comprises about 20 amino acids in two short -helical segments, each seven to nine amino acid residues long, separated by a β turn (Fig. 4). This structure generally is not stable by itself; it is simply the reactive portion of a somewhat larger DNA-binding domain. One of the two-helical segments is called the recognition helix, because it usually contains many of the amino acids that interact with the DNA in a sequence-specific way. This α helix is stacked on other segments of the protein structure so that it protrudes from the protein surface. When bound to DNA, the recognition helix is positioned in or nearly in the major groove. The Lac repressor has this DNA-binding motif (Fig. 4).
FIGURE 4 Helix-turn-helix. (a) DNA-binding domain of the Lac repressor (PDB ID 1LCC). The helix-turn-helix motif is shown in red and orange; the DNA recognition helix is red. (b) Entire Lac repressor (derived from PDB ID 1LBG). The DNA-binding domains are gray, and the α helices involved in tetramerization are red. The remainder of the protein (shades of green) has the binding sites for allolactose. The allolactose-binding domains are linked to the DNA-binding do mains through linker helices (yellow). (c) Surface rendering of the DNA-binding domain of the Lac repressor (gray) bound to DNA (blue). (d) The same DNA-binding domain as in (c), but separated from the DNA, with the binding interaction surfaces shown. Some groups on the protein and DNA that interact through hydrogen-bonding are shown in red; some groups that interact through hydrophobic inter actions are in orange. This model shows only a few of the groups in volved in sequence recognition. The complementary nature of the two surfaces is evident.
Zinc Finger In a zinc finger, about 30 amino acid residues form an elongated loop held together at the base by a single Zn2+ ion, which is coordinated to four of the residues (four Cys, or two Cys and two His). The zinc does not itself interact with DNA; rather, the coordination of zinc with the amino acid residues stabilizes this small structural motif. Several hydrophobic side chains in the core of the structure also lend stability. Figure 5 shows the interaction between DNA and three zinc fingers of a single polypeptide from the mouse regulatory protein Zif268.
Many eukaryotic DNA-binding proteins contain zinc fingers. The interaction of a single zinc finger with DNA is typically weak, and many DNA-binding proteins, like Zif268, have multiple zinc fingers that substantially en hance binding by interacting simultaneously with the DNA. One DNA-binding protein of the frog Xenopus has 37 zinc fingers. There are few known examples of the zinc finger motif in prokaryotic proteins. The precise manner in which proteins with zinc fingers bind to DNA differs from one protein to the next. Some zinc fingers contain the amino acid residues that are important in sequence discrimination, whereas others appear to bind DNA nonspecifically (the amino acids required for specificity are located elsewhere in the protein). Zinc fingers can also function as RNA-binding motifs—for example, in certain proteins that bind eu karyotic mRNAs and act as translational repressors. We discuss this role later (Section 28.3).
Homeodomain Another type of DNA-binding domain has been identified in a number of proteins that function as transcriptional regulators, especially during eukaryotic development. This domain of 60 amino acids—called the homeodomain, because it was discovered in homeotic genes (genes that regulate the development of body pat terns)—is highly conserved and has now been identified in proteins from a wide variety of organisms, including humans (Fig. 6). The DNA-binding segment of the domain is related to the helix-turn-helix motif. The DNA sequence that encodes this domain is known as the homeobox.
FIGURE 5 Zinc fingers. Three zinc fingers (gray) of the regulatory protein Zif268, complexed with DNA (blue and white) (PDB ID 1A1L). Each Zn2+ (maroon) coordinates with two His and two Cys residues (not shown).
FIGURE 6 Homeodomain. Shown here is a homeodomain bound to DNA; one of the helices (red), stacked on two others, can be seen protruding into the major groove (PDB ID 1B8I). This is only a small part of the much larger protein Ultrabithorax (Ubx), active in the regulation of development in fruit flies.
