How do proteins that turn genes on and off recognize their sites of action within the genome? [HN1], [HN2], [HN3], [HN4] Lock-and-key type molecular complementarity between a regulatory protein and its DNA binding site provides the primary recognition. [HN5] A constellation of electrostatic and hydrophobic interactions between matching surfaces of the DNA helix and the protein establish high-affinity and sequence-specific binding. The effectiveness of this macromolecular matchmaking, first elucidated by elegant experiments in prokaryotes, is challenged by the complexities of eukaryotes. There are hundreds of regulatory transcription factors that function by binding DNA sequences within their target genes. Almost all of these proteins are encoded by multigene families. Members of a family display the same structural fold for binding DNA and recognize similar DNA sequences. How can specificity be obtained within such a complex world?
Combinatorial arrays of multiple proteins add specificity and stability to DNA-protein interactions. Homodimers and heterodimers can be formed between members of the same gene family. Alternatively, partnerships can form between two proteins that belong to unrelated groupings. Wolberger and colleagues [HN6] have studied one such partnership (1), and their report on page 1037 provides a snapshot of the molecular basis of combinatorial control of transcription. The report describes the crystal structure of the ternary complex of GABPa, a member of the ets gene family, with its heterotypic partner GABPb on a DNA duplex.
The ets gene family dramatically illustrates the specificity problem (2). ets genes are present in all metazoan phyla, [HN7] with more than 20 homologs in the human genome. The ETS domain, a highly conserved 85-amino acid region, defines the family and directs DNA binding. The DNA binding sites of all ets proteins include the core recognition sequence 5¢-GGA-3¢. Additional DNA contacts that also require conserved sequences extend the binding site to include at least nine base pairs. With such a high degree of conservation, how is specificity programmed into the family? For example, GABP is ubiquitous in mammalian systems and regulates expression of respiratory and translational machinery genes. The wide-spread distribution of GABP overlaps that of many tissue-restricted ets gene products. What determines that GABP regulates only its target genes?
One answer comes from the versatility of the ETS domain-DNA interaction. The winged helix-turn-helix motif of the ETS domain is composed of three a helices and a four-stranded b sheet (1, 3-5). In the GABPa structure, helix 3 of the helix-turn-helix motif binds DNA in the major groove with two invariant arginines hydrogen bonding directly to the guanine residues of the GGA core. Other structural elements, including the b sheet and helix 1, make phosphate contacts on each flank of the GGA core, and these contacts indirectly specify additional sequence preferences (see the figure). The phosphate contacts made by GABPa are similar, but not identical, to those reported in the crystal structure of PU.1 with DNA (5). Furthermore, the structures of PU.1- and GABP-DNA complexes display slightly different bonding networks between helix 3 and the GGA motif. Indeed, PU.1 is known to bind an alternative core, AGA, and this difference can be explained by the structural differences at the DNA-protein interface. How these and other subtle variations in DNA recognition can determine specificity within the ets family can now be investigated.
ILLUSTRATION: K. SUTLIFF
Specific association with GABPb further regulates the DNA binding specificity of GABPa. GABPb interacts with GABPa through four ankyrin repeats. Each repeat is composed of two a helices in a coiled-coil configuration with an intervening loop punctuated by a b turn at its tip (1, 6). Each tip interacts with a distinct part of GABPa, including parts of the ETS domain and the carboxyl-terminal flanking region, which includes helices 4 and 5. This heterotypic interface illustrates the diversity of structural coupling between interacting proteins while emphasizing the need for multiple contacts to mediate stable and specific interactions.
Insights from the GABP-DNA structure extend beyond the static picture of the complex. The GABPa-GABPb ternary complex is 100 times as stable as a binary complex formed only with GABPa. However, GABPb does not directly contact DNA. Instead, intermolecular interactions between the two subunits indirectly affect DNA binding. First, a lysine within the third ankyrin repeat hydrogen bonds with a glutamine of GABPa that directly contacts DNA. This glutamine contacts a single phosphate in concert with a backbone amide of a leucine at the amino-terminus of helix 1. Three other helix 1-ankyrin repeat interactions also are detected. This structural coupling suggests that GABPb buttresses the helix 1-DNA interaction and that this single phosphate contact is critical for stable DNA binding.
An interaction between the first ankyrin repeat of GABPb and helix 5 of GABPa is a second potential effector of enhanced DNA binding. The authors speculate that helix 5 is functionally analogous to helix 4 of Ets-1, which also lies on the carboxyl-terminal side of the ETS domain (4). In Ets-1, helix 4 negatively regulates DNA binding (7). In the absence of DNA, this helix packs against helix 1 as well as against two additional helices that lie amino-terminal to the ETS domain. This helical packing is inhibitory and must be disrupted during DNA binding (8). In the GABPa-GABPb complex, helix 5 does not contact helix 1. The authors propose that GABPb alters the position of helix 5, derepressing DNA binding. This model predicts that helix 5 inhibits the DNA binding of GABPa alone and that helix 5 will be positioned differently in the absence of GABPb, predictions that can now be tested.
Optimal DNA binding by GABP requires more than the ETS domain-ankyrin repeat interaction. A leucine zipper motif [HN8] within GABPb, which is not present in the crystal structure, directs formation of a heterotetramer (a2-b2). In this configuration GABP recognizes two GGA sites and displays even higher DNA binding affinity (9) (see the figure). Among the ets proteins, GABPa is the only one that binds DNA as an oligomer. Thus, GABPb acts at several levels to add specificity and affinity to the DNA binding activity of GABPa.
Future studies should address whether other ets proteins are regulated by analogous partners. GABPb apparently exhibits a high degree of specificity for GABPa, as ternary complexes with other ets proteins have not been detected (10). There are a plethora of candidate partners for other ets proteins. The report by Wolberger and colleagues (1) provides a structural and mechanistic framework for understanding these partnerships (for example, positioning helix 1 of the ETS domain as well as counteracting inhibitory sequences that lie outside of the ETS domain).
DNA binding cooperativity is frequently reported between transcription factors that function at a single promoter. Together with biochemical studies of other ets proteins, the GABP structure suggests that autoinhibition, conformational change and allosteric effects are potent strategies for modulating the affinity and specificity of DNA-protein interactions and derepressing autoinhibition. Just as transcription is controlled both by positive- and negative-acting proteins, transcription factors can be themselves regulated by opposing pathways.
Muscle-Specific Regulation of Transcription: A Catalog of Regulatory Elements by Laura L. L-pez and James W. Fickett presents a summary of published information on muscle-specific transcriptional regulation.
Pedro's BioMolecular Research Tools is a collection of WWW links to information and services useful to molecular biologists. It provides links to molecular biology search and analysis tools; bibliographic, text, and Web search services; guides and tutorials; and biological and biochemical journals and newsletters.
The World Wide Web Virtual Library: Biosciences points to virtual library pages for Biomolecules, and Biochemistry and Molecular Biology. Each of these pages presents a long list of Web resources. The World Wide Web Virtual Library Biomolecules covers molecular sequence and structure databases, metabolic pathway databases, and other lists of Web resources. The World Wide Web Virtual Library: Biochemistry and Molecular Biology is a list of resources listed by provider.
Cell & Molecular Biology Online is a well-organized list of Web resources for cell and molecular biologists. For each resource, a brief description is provided.
CSUBIOWEB, the California State University Biological Sciences Web server, provides links to other Web sites on cell biology and molecular biology.
The Dictionary of Cell Biology (London: Academic Press, 1995) defines transcription, leucine zipper, and other terms used in this research commentary.
Biotech Life Science Dictionary is a free resource that defines terms in biochemistry, biotechnology, botany, cell biology, and genetics, including terms used in this research commentary.
Protein Synthesis is a tutorial on the processes involved in Protein Synthesis, starting from the genetic information in DNA, through transcription to produce messenger RNA, and translation of mRNA to a polypeptide. This tutorial is a section of Principles of Protein Structure Using the Internet, a Birkbeck College (University of London) accredited Advanced Certificate course.
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The Structure of GABP / :
An ETS Domain- Ankyrin Repeat Heterodimer Bound to DNAScience 13 February 1998; 279 (5353):1037 (in Reports) A. H. Batchelor, D. E. Piper, F. C. de la Brousse, S. L. McKnight, C. Wolberger |
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Copyright © 1998 by the American Association for the Advancement of Science.