Weak Bonds Correctly Position Proteins Along Dna And Rna Molecules

FIGURE 5-19 Protein-single-strand DMA interaction for single-Strand DNA-binding protein (SSB). S5B »5 shown in gray and single-stranded DMA ts shown in red. {Raghcmathdn S., Kozlov AG., Lehman TM, and Waksrrwi C. 2000. Nature Structural Btology, 8: 6A8.) (mage prepared with MotScript, BobSaipt, and Raster 30.

FIGURE 5-19 Protein-single-strand DMA interaction for single-Strand DNA-binding protein (SSB). S5B »5 shown in gray and single-stranded DMA ts shown in red. {Raghcmathdn S., Kozlov AG., Lehman TM, and Waksrrwi C. 2000. Nature Structural Btology, 8: 6A8.) (mage prepared with MotScript, BobSaipt, and Raster 30.

DNA-binding proteins mediate many of the central processes in biology. The bonds that hold these proteins onto DNA are the same collection of weak bonds that give proteins, DNA, and RNA their own specific three-dimensional configurations. The most abundant DNA-binding proteins have a structural roie in packaging and compacting the huge amount of DNA thai must be fitted into the cell. For example, the nucleus of a human cell is only 10 urn (ltr5 meter) across but contains roughly 1 meters of double-stranded DNA.

There are many ways that proteins can recognize DNA. Some protein-DNA interactions are specific for particular sequences of DNA, whereas others are more specific for DNA in specific conformations. For example, when DNA is unwound in the cell during DNA replication or recombination, the single strands are rapidly bound by single-stranded DNA-binding proteins (SSBs}. These proteins bind with little sequence specificity but are highly specific foT single-versus double-stranded DNA. To accomplish this specificity, the primary interactions between SSBs and the single-stranded DNA are through ionic or hydrogen bond interactions with the phosphate backbone or through intercalation of bulky ring-shaped sale chains (for example, Tyr or Trp) between the bases (Figure 5-19).

Most DNA-binding proteins wo will consider in this book recognize specific DNA sequences in double-stranded DNA. Such proteins are frequently involved in choosing specific sequences in the genome to act as sites for the initiation of transcription or replication, or other DNA transactions. Indeed, 2—3% of prokaryotic proteins and 6—7% of eukaryotic proteins are either known or predicted to be sequence-specific DNA-binding proteins. By far the most common mechanism for protein recognition of a specific DNA sequence is through the insertion of an a helix in the so-called major groove of the DNA (see Figure 5-20), As was evident in Figure 5-2 and is shown explicitly in Figure 6-1, the double helix has a wide groove known as the major groove and a narrow, or minor groove. Recognition using an a helix that inserts in the major groove is advantageous for several reasons.

1. The width and depth of the major groove is a very good match to the dimensions of an a helix. This match allows weak interactions to occur between the DNA and approximately half of the surface of the a helix,

2. The major groove is rich in hydrogen bond acceptors and dnnors located on the edges of the bases (see Figure 6-10). More importantly, the pattern of hydrogen bonding elements is distinct for each of the base pairs. This allows the pattern of hydrogen bond donors and acceptors to act as a code for the sequence of the DNA, in the same way that hydrogen bonding between the base pairs ensures the appropriate recognition of complementary DNA

WeoA. Bonds Correctly Position Protei m along DNA and RNA Molecules as sequences during DNA hybridization, A diagram of the pattern of hydrogen-bonding donor and acceptor residues in the major groove for each base pair illustrates the distinct pattern for each base pair [see Figure 6-10). Note that not only can a G:C base pair be easily distinguished from an A:T base pair, but A:T and T:A. and G:C and C:G base pairs can also be distinguished. In contrast, the pattern of base pairs in the minor groove has significantly less information and generally only allows the distinction of A:T and G:C. 3. a helices have a dipole moment that leads to their N-terminal end being positively charged. This positively-charged end frequently makes weak interactions with the phosphate backbone adjacent to the major groove.

The helfx-turn-helix motif was the first protein motif involved in sequence-specific DNA binding to be identified. This motif is composed of two adjacent u helices that are separated by a short turn (Figure 5-21). One a helix, called the recognition helix, is responsible for DNA recognition. The second a helix is located approximately perpendicular to the first a helix. Although these two helices form the core of the DNA recognition motif, other nearby regions of helix-turn-helix DNA-binding proteins frequently stabilize the arrangement of these two a helices and contact the DNA. Other DNA-binding motifs also insert n helices into the major groove, such as the zinc finger and leucine zipper DNA-binding motifs (as we shall discuss in Chapter 17).

Whereas the use of an a helix is the predominant form of specific DNA recognition, some proteins do use different strategies. An extreme example of this is seen with the TATA-binding protein (TBP), which determines the site of transcriptional initiation at many eukaryotic promoters (see Chapter 12). TBP uses an extensive region of p sheet to recognize the minor groove of the so-called TATA-box (Figure 5-22). So, in this case, we see the use of p sheet instead of « helix and interactions with the minor groove rather than the major groove (for a detailed discussion of this matter, see Chapter 12).

Proteins Scan along DNA to Locate a Specific DNA'Binding Site

Many DNA-binding proteins make substantia] contacts with the DNA backbone as well as with the specific tiase pairs of their recognition sites, Mediating these backbone contacts are patches of positively-charged amino acids located at sites very close to those that bind to the base pairs. These associations rely primarily on electrostatic attraction between these positive patches and the negatively-charged phosphate backbone of the DNA. Because the backbone has a similar negatively-charged surface, regardless of the sequence, these protein-DNA backbone contacts contribute substantially both the specific and nonspecific affinity of a protein for DNA, Thus, even a highly specific DNA-binding protein will have a substantial affinity for nonspecific DNA sites as Well.

For example, the affinity of some well-characterized regulators of gene expression (such as the lactose repressor) for their recognition sequences is about ID5-fold greater than their affinity for nonspecific DNA. As a consequence, in the cell these proteins are typically bound at a number of nonspecific sites as well as at their specific target sequence. This is due to the much larger number of nonspecific sites compared to the specific sites. Indeed, every nucleotide in the genome

f 1 CURE 5-20 Schematic of interaction between the recognition helix of X repressor monomer and major groove of operator DNA. (Sourte : Adapted from Jordan S.ft. end t'abo CO. Î988. Structure of the lambda complex at 2.5 A resolution Science 342 893-899. Copyn^it © 1988 American Association for the Advancement ot Science Used with permission.)

f 1 CURE 5-20 Schematic of interaction between the recognition helix of X repressor monomer and major groove of operator DNA. (Sourte : Adapted from Jordan S.ft. end t'abo CO. Î988. Structure of the lambda complex at 2.5 A resolution Science 342 893-899. Copyn^it © 1988 American Association for the Advancement ot Science Used with permission.)

FIGURE 5-21 Geometry of \ repressoroperator complex. The schematic shows two monomers of A repressor bound to the operator The helices in each monomer are ta-beied t to 5, tt (5 he Ha 3 whfefl inserts iritotlto major groove as shown in Figure 5-20 (Source Adapted from Jordan 5.R and Pabo C.O 198B. Structure of the lambda complex at A [isolation Science 24? 893-899, f. 2b, page 895. Copyright i£: 1988 American Association for the Advancement of Science Used with permission.)

FIGURE 5-21 Geometry of \ repressoroperator complex. The schematic shows two monomers of A repressor bound to the operator The helices in each monomer are ta-beied t to 5, tt (5 he Ha 3 whfefl inserts iritotlto major groove as shown in Figure 5-20 (Source Adapted from Jordan 5.R and Pabo C.O 198B. Structure of the lambda complex at A [isolation Science 24? 893-899, f. 2b, page 895. Copyright i£: 1988 American Association for the Advancement of Science Used with permission.)

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