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Protein-nucleic acid interactions Editorial overview
Protein-nucleic acid interactions Editorial overview Simon E.V. Phillips University of Leeds, Leeds, UK Current Opinion in Structural Biology 1992, 2:69-70 Proteins that interact with nucleic acids have a key role to play in all biological organisms. They are necessary for the control of expression of genetic information, and its replication, packaging and protection from possibly hostile environments. Knowledge of the three-dimensional structures of such proteins is essential to full understanding of their functions, and there has been a large increase in crystallographic and NMR structure determinations during the past few years. In particular, developments in chemical synthesis and purification of oligonucleotides have led to an increasing number of published structures for protein-DNA complexes, and we can expect this to extend to protein-RNA complexes during the next few years. During 1991, dramatic progress was made in a number of areas, and several other important structures are waiting in the wings to appear in the coming year. The first seven reviews cover the structure of duplex DNA and the proteins that recognize it. Later reviews cover proteins that recognize single-stranded DNA, RNA and finally virus structures. During the past year, the appearance of two high-resolution structures of complexes, Escherichia coli catabolite gene activator protein (CAP) and EcoRV restriction endonuclease, has finally laid a strong structural foundation for understanding such bending. In both cases, some base steps are completely unstacked. The helical parameters of duplex DNA in known complex structures continue to mimic the expected values for relaxed B-DNA in solution rather better than do those for oligonucleotitte crystal structures. This suggests that complex formation stabilizes the DNA conformation, rendering it more resistant to distortion by crystal lattice forces. The general principle of sequence specificity mediated by direct interactions between protein side chains and nucleic acid bases continues to hold good for the majority of cases. These interactions often involve regions of secondary structure in the protein but the importance of flexible loops and arms is becoming increasingly apparent. The details of the latter interactions are very important and can only be discerned with confidence where structures are determined at high resolution and are carefully refined, a-Helices continue to be the secondary structure elements most often found docked into the major groove of B-DNA.
In the first review, Travers (pp 71-77) considers progress made in understanding the conformation adopted by DNA in complexes with proteins, and to what extent this reflects the properties of particular base sequences. He takes the view that conformations intermediate between A- and B-DNA do exist, an issue currently under debate, and argues that the DNA in the crystal structure of the Zif268 complex is an example. The problem is partly semantic, however, and depends on which geometrical parameters are used. He discusses a number of recent crystal structures of protein-DNA complexes that show bending to a greater and lesser extent and demonstrate a dependence of binding affinity on pre-existing conformation or inherent flexibility of particular base sequences. He then addresses the biological effects of bending, especially transcriptional activation and the action of CAP and the factor for inversion stimulation (Fis). Baldwin (pp78-83) reviews progress on nucleosome structure, a standard model for sequence-dependent DNA conformational preferences. Whereas the dispute over the shapes of the histone octamer and nucleosome core particles is finally being resolved, work on the highresolution structure of the core particle still progresses slowly. Crystals of the globular domain of histone H5 protein have been prepared, however, and diffract to high resolution. Prominent over the past year were studies of high mobility group proteins, together with suggestions that they may be general transcription factors. Comprehensive reviews by Suck (pp 84-92) and Winkler (pp 93-99) cover advances in the structure and function of nucleases and restriction endonucleases, respectively. The former describes new insights into the catalytic function of several well known structures, gleaned from new binding and site-directed mutagenesis studies. Proposed mechanisms frequently, but not universally, involve attack by metal-activated water molecules. Also reviewed are several new nuclease structures, including RNase H and P1 nuclease, the latter having a polypeptide fold and a cluster of three zinc ions which are very like those of phospholipase C. The second restriction endonuclease structure determined, EcoRV, is reviewed and compared with that of EcoRI; the endonucleases exhibit surprising similarities in active-site structure despite having unrelated sequences and completely different folds. EcoRV is remarkable in that its ~ t y for random-sequence DNA
Abbreviations CAP~atabolite gene activator protein; Fis---factor for inversion stimulation; RNP--ribonucleoprotein. (~) Current Biology Ltd ISSN 0959-440X
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Protein-nucleic acid interactions
is similar to that for its recognition site, all of its selectivity being mediated by catalytic efficiency (i.e. kcat alone). Both EccRI and EcoRV complexes with cognate sites show distorted DNA, which is kinked in the former and strongly bent in the latter, especially at the cleavage sites. A non-specific EcoRV complex, however, binds essentially undistorted B-DNA. Three reviews describe progress with commonly occurring DNA-binding motifs: the helix-turn-helix (Brennan, ppl00-108), zinc fingers (Kaptein, pp109-115) and the leucine zipper (Pathak and Sigler, pp 116-123). Several new structures of helix-turn-helix proteins have appeared, including the helix-bending Fis protein, and a complex of CAP in which the predicted bending was observed directly. In the absence of DNA, Fis has disordered regions at chain termini, a property that it shares with a number of repressor molecules. High-resolution (1.8A) low-temperature studies of the L-repressor--operator complex have shown that the flexible arms of X repressor make highly specific contacts to the DNA, which were not revealed by earlier work at 2.5 A. A structure has also appeared for the 434--Cro complex. Structures for homeodomain complexes seem to be leading to a general model for their interactions with DNA. Progress on zinc fingers has been dramatic, with DNA complex structures reported for the three fingers of 7_M268 and the glucocorticoid receptor DNA-binding domain. Zif268 follows the major groove, each finger interacting essentially identically with the DNA, creating motifs with a period of 3 bp. The glucocorticoid receptor fold is completely different - - the dimeric molecule docks to its symmetric DNA binding site rather like a bacterial repressor. Both proteins, however, have at-helices lodged in the major groove, with side chains making direct contacts to the base-pair edges. GAL4 has been shown to have a binuclear zinc cluster; a full three-dimensional structure determination is in progress. Although no natural leucine-zipper protein structures have been reported, the structure of a short synthetic peptide corresponding to the 'zipper' region of GCN4 has now been determined crystallographically. Pathak and Sigler (pp 116-123) take care to remind us of the history of the helical coiled-coil structure, which is all the zipper structure actually is. One cannot fail to be impressed, however, by the subtlety of the sequence variations that is found in these proteins and which is used to control dimerization and stability in such a deceptively simple structure. Single-stranded DNA-binding proteins represent a ne• glected area, but a review by Kneale (pp 124-130) shows that developments are on the way. The one well known crystallographic structure, fd gene 5 protein, is not totally consistent with NMR and other data, and further work is in progress. The crystal structure of E. coli Rec A has now also been published. E. coli SSB, T4 gene 32 and RegA proteins all have interesting properties, and structural studies of these proteins are under way in several laboratories. In the meantime, biophysical and mutagenesis data are accumulating.
In two reviews on protein-RNA interactions, Nagai (pp 131-137) considers the field in general whereas Moras (pp 138-142) concentrates on tRNA-aminoacyL tRNA synthetase complexes. Although the difference between the chemical etructures of RNA and DNA is small, RNA shows a much wider variation in tertiary structure, including fully folded globular molecules such as tRNA. This allows for a great variety of possible modes of interactions of RNAwith proteins. Nagai describes the bubble, bulge, stem/loop and pseudoknot RNA folds, and gives examples of proteins that bind to them. The crystal structures has been determined for the ribonucleoprotein (RNP) domain from U1 A, a component of U1 small nuclear RNP, a protein which recognizes an RNA stern/loop. It consists of two m-helices and a four-stranded antiparaUel [3-sheet, the central two strands of which carry the conserved RNP1 and RNP2 sequence motifs. Chemical modification and mutagenesis experiments have identified important residues, and have allowed a model of the protein-RNA complex to be built, but no experimental structure determination has been reported. Aminoacyl-tRNA synthetases are classified into two groups; until recently, structures were known for three class-I enzymes (one in a complex with tRNA) but only one class-II enzyme. The crystal structure determination of aspartyl-tRNA synthetase (class II) in a complex with its cognate tRNA has now been published; this has n o t only provided information on tRNA recognition for classII enzymes but has also allowed comparison of their conserved structural features. Interestingly, class-I and class-II synthetases approach the tRNA acceptor stem from opposite sides in their respective complexes. High-resolution refinement of the glutaminyt-tRNA synthetase complex (class I) has now revealed the details of the anticodon interactions; the three bases are splayed out and each lies in its own pocket on the enzyme surface. The structure of the free tRNA is unknown but it seems likely the anticodon structure is different from that in the complex, judging by other known tRNA structures. The sequence for cysteinyl-tRNA synthetase has now been published, identifying it as belonging to class I, and completing the set of aminoacyt-tRNA synthetase sequences for aU 20 common amino acids. When the first few structures of the icosahedral viruses were determined, there were great hopes that it might be possible to observe the nucleic acid in electron-density maps, in addition to the protein capsid, but it was always disordered with respect to the crystal lattice and, hence, invisible. This situation has now changed and a number of recent structures show evidence of ordered nucleic acid, as described by Stockley (pp 143-149) in the final review. In the canine parvovirus and phage ~X174 structures, for example, over 10% of the viral DNA is at least partially ordered. High-resolution fibre diffraction studies of tobacco mosaic virus have also revealed details of the RNA structure. Knowledge of the interactions between the nucleic acid and protein coat is very valuable in understanding viral assembly, and such observations should result in significant progress in this field. SEV Phillips, Department of Biochemistry and Molecular Biology, University of Leads, Leeds LS2 9Tr, UK.
In the first review, Travers (pp 71-77) considers progress made in understanding the conformation adopted by DNA in complexes with proteins, and to what extent this reflects the properties of particular base sequences. He takes the view that conformations intermediate between A- and B-DNA do exist, an issue currently under debate, and argues that the DNA in the crystal structure of the Zif268 complex is an example. The problem is partly semantic, however, and depends on which geometrical parameters are used. He discusses a number of recent crystal structures of protein-DNA complexes that show bending to a greater and lesser extent and demonstrate a dependence of binding affinity on pre-existing conformation or inherent flexibility of particular base sequences. He then addresses the biological effects of bending, especially transcriptional activation and the action of CAP and the factor for inversion stimulation (Fis). Baldwin (pp78-83) reviews progress on nucleosome structure, a standard model for sequence-dependent DNA conformational preferences. Whereas the dispute over the shapes of the histone octamer and nucleosome core particles is finally being resolved, work on the highresolution structure of the core particle still progresses slowly. Crystals of the globular domain of histone H5 protein have been prepared, however, and diffract to high resolution. Prominent over the past year were studies of high mobility group proteins, together with suggestions that they may be general transcription factors. Comprehensive reviews by Suck (pp 84-92) and Winkler (pp 93-99) cover advances in the structure and function of nucleases and restriction endonucleases, respectively. The former describes new insights into the catalytic function of several well known structures, gleaned from new binding and site-directed mutagenesis studies. Proposed mechanisms frequently, but not universally, involve attack by metal-activated water molecules. Also reviewed are several new nuclease structures, including RNase H and P1 nuclease, the latter having a polypeptide fold and a cluster of three zinc ions which are very like those of phospholipase C. The second restriction endonuclease structure determined, EcoRV, is reviewed and compared with that of EcoRI; the endonucleases exhibit surprising similarities in active-site structure despite having unrelated sequences and completely different folds. EcoRV is remarkable in that its ~ t y for random-sequence DNA
Abbreviations CAP~atabolite gene activator protein; Fis---factor for inversion stimulation; RNP--ribonucleoprotein. (~) Current Biology Ltd ISSN 0959-440X
69
70
Protein-nucleic acid interactions
is similar to that for its recognition site, all of its selectivity being mediated by catalytic efficiency (i.e. kcat alone). Both EccRI and EcoRV complexes with cognate sites show distorted DNA, which is kinked in the former and strongly bent in the latter, especially at the cleavage sites. A non-specific EcoRV complex, however, binds essentially undistorted B-DNA. Three reviews describe progress with commonly occurring DNA-binding motifs: the helix-turn-helix (Brennan, ppl00-108), zinc fingers (Kaptein, pp109-115) and the leucine zipper (Pathak and Sigler, pp 116-123). Several new structures of helix-turn-helix proteins have appeared, including the helix-bending Fis protein, and a complex of CAP in which the predicted bending was observed directly. In the absence of DNA, Fis has disordered regions at chain termini, a property that it shares with a number of repressor molecules. High-resolution (1.8A) low-temperature studies of the L-repressor--operator complex have shown that the flexible arms of X repressor make highly specific contacts to the DNA, which were not revealed by earlier work at 2.5 A. A structure has also appeared for the 434--Cro complex. Structures for homeodomain complexes seem to be leading to a general model for their interactions with DNA. Progress on zinc fingers has been dramatic, with DNA complex structures reported for the three fingers of 7_M268 and the glucocorticoid receptor DNA-binding domain. Zif268 follows the major groove, each finger interacting essentially identically with the DNA, creating motifs with a period of 3 bp. The glucocorticoid receptor fold is completely different - - the dimeric molecule docks to its symmetric DNA binding site rather like a bacterial repressor. Both proteins, however, have at-helices lodged in the major groove, with side chains making direct contacts to the base-pair edges. GAL4 has been shown to have a binuclear zinc cluster; a full three-dimensional structure determination is in progress. Although no natural leucine-zipper protein structures have been reported, the structure of a short synthetic peptide corresponding to the 'zipper' region of GCN4 has now been determined crystallographically. Pathak and Sigler (pp 116-123) take care to remind us of the history of the helical coiled-coil structure, which is all the zipper structure actually is. One cannot fail to be impressed, however, by the subtlety of the sequence variations that is found in these proteins and which is used to control dimerization and stability in such a deceptively simple structure. Single-stranded DNA-binding proteins represent a ne• glected area, but a review by Kneale (pp 124-130) shows that developments are on the way. The one well known crystallographic structure, fd gene 5 protein, is not totally consistent with NMR and other data, and further work is in progress. The crystal structure of E. coli Rec A has now also been published. E. coli SSB, T4 gene 32 and RegA proteins all have interesting properties, and structural studies of these proteins are under way in several laboratories. In the meantime, biophysical and mutagenesis data are accumulating.
In two reviews on protein-RNA interactions, Nagai (pp 131-137) considers the field in general whereas Moras (pp 138-142) concentrates on tRNA-aminoacyL tRNA synthetase complexes. Although the difference between the chemical etructures of RNA and DNA is small, RNA shows a much wider variation in tertiary structure, including fully folded globular molecules such as tRNA. This allows for a great variety of possible modes of interactions of RNAwith proteins. Nagai describes the bubble, bulge, stem/loop and pseudoknot RNA folds, and gives examples of proteins that bind to them. The crystal structures has been determined for the ribonucleoprotein (RNP) domain from U1 A, a component of U1 small nuclear RNP, a protein which recognizes an RNA stern/loop. It consists of two m-helices and a four-stranded antiparaUel [3-sheet, the central two strands of which carry the conserved RNP1 and RNP2 sequence motifs. Chemical modification and mutagenesis experiments have identified important residues, and have allowed a model of the protein-RNA complex to be built, but no experimental structure determination has been reported. Aminoacyl-tRNA synthetases are classified into two groups; until recently, structures were known for three class-I enzymes (one in a complex with tRNA) but only one class-II enzyme. The crystal structure determination of aspartyl-tRNA synthetase (class II) in a complex with its cognate tRNA has now been published; this has n o t only provided information on tRNA recognition for classII enzymes but has also allowed comparison of their conserved structural features. Interestingly, class-I and class-II synthetases approach the tRNA acceptor stem from opposite sides in their respective complexes. High-resolution refinement of the glutaminyt-tRNA synthetase complex (class I) has now revealed the details of the anticodon interactions; the three bases are splayed out and each lies in its own pocket on the enzyme surface. The structure of the free tRNA is unknown but it seems likely the anticodon structure is different from that in the complex, judging by other known tRNA structures. The sequence for cysteinyl-tRNA synthetase has now been published, identifying it as belonging to class I, and completing the set of aminoacyt-tRNA synthetase sequences for aU 20 common amino acids. When the first few structures of the icosahedral viruses were determined, there were great hopes that it might be possible to observe the nucleic acid in electron-density maps, in addition to the protein capsid, but it was always disordered with respect to the crystal lattice and, hence, invisible. This situation has now changed and a number of recent structures show evidence of ordered nucleic acid, as described by Stockley (pp 143-149) in the final review. In the canine parvovirus and phage ~X174 structures, for example, over 10% of the viral DNA is at least partially ordered. High-resolution fibre diffraction studies of tobacco mosaic virus have also revealed details of the RNA structure. Knowledge of the interactions between the nucleic acid and protein coat is very valuable in understanding viral assembly, and such observations should result in significant progress in this field. SEV Phillips, Department of Biochemistry and Molecular Biology, University of Leads, Leeds LS2 9Tr, UK.