DNA-Protein Interactions DNA-Protein Interactions Andrew Travers MRC Laboratory of Molecular Biology Cambridge UK I~nl SPRINGER-SCIENCE+BUSINESS MEDIA, B.v. Firstedition 1993 Reprinted 1994 © 1993 Springer Science+Busin.:ss Media Dordredtt Originally publisbed by Chapman & Hali in 1993 Typeset in 10/12 pt Palatino by Expo Holdings, Malaysia ISBN 978-0-412-25990-6 Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the UK Copyright Designs and Patents Act, 1988, this publication may not be reproduced, stored, or transmitted, in any form or by any means, without the prior permission in writing of the publishers, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to the publishers at the London address printed on this page. The publisher makes no representation, express or implied, with regard to the accuracy of the information contained in this book and cannot accept any legal responsibility or liability for any errors or omissions that may be made. A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication data Travers, A. A. (Andrew Arthur) DNA-Protein Interactionsl Andrew Travers. p. cm. Includes bibliographical references and index. ISBN 978-0-412-25990-6 ISBN 978-94-011-1480-6 (eBook) DOI 1O.1007/978-94-011-1480-6 1. DNA-Protein Interactions. 1. Title. QP624. 75. P74T7 1993 574.87'3282-dc20 92-38467 CIP 8 Printed on permanent acid-free text paper, manufactured in acrordance with the proposed ANSIINISO Z 39. 48-199X and ANSI Z 39.48-1984 Contents Preface ix 1 DNA structure 1 1.1 Structural features of DNA 1 1.2 DNA polymorphism 3 1.3 Conformational variability of DNA 4 1.4 Intrinsic bending of DNA 8 1.5 DNA supercoiling and topology 12 1.6 The topology of protein-bound DNA 18 1.7 Structure of supercoiled DNA 20 References 26 2 DNA-protein interactions: The three-dimensional architecture of DNA-protein complexes 28 2.1 General principles 28 2.2 Local DNA conformation and protein binding 29 2.3 DNA wrapping 34 2.4 DNA configuration and sequence periodicity 43 2.5 The establishment of DNA architecture 44 References 50 3 DNA-protein interactions: Sequence specific recognition 52 3.1 General principles of sequence specific recognition 52 3.2 Structural motifs for sequence specific binding 53 3.2.1 The helix-tum-helix motif 53 Cl and cra repressors 56 Catabolite activator protein (CAP) 59 lac repressor protein 60 Tryptophan repressor 62 vi Contents 3.2.2 Zinc-containing DNA-binding domains 64 The interaction of zinc fingers with DNA 69 3.2.3 Other DNA-binding structures 74 3.2.4 Heterodimer formation 76 3.3 Co-operative binding to DNA 80 3.4 Co-operativity at a distance: DNA looping 82 3.5 Protein flexibility in DNA-protein complexes 85 References 86 4 The mechanism of RNA chain initiation 87 4.1 The promoter 87 4.2 RNA polymerases' 88 4.3 The kinetics of transcription initiation 90 4.4 The molecular interactions of RNA polymerase with a promoter site 91 4.5 The topological consequences of transcription 96 4.6 Transcriptional activators 99 4.7 Transcriptional activation by negative superhelicity 104 4.8 How does RNA polymerase melt promoter DNA? 105 References 108 5 The regulation of promoter selectivity in eubacteria 109 5.1 Control of stable RNA synthesis 109 5.2 Stable RNA promoters 111 5.3 The transition from exponential growth to stationary phase and vice versa 115 5.4 Sigma factors and promoter recognition 118 5.5 The heat shock response 119 5.6 0.54: A target for transcriptional enhancers 120 5.7 The transcriptional programmes of bacterial viruses 125 References 128 6 The mechanism of eukaryotic transcription 130 6.1 Eukaryotic transcriptional regulatory elements 133 6.2 Eukaryotic transcriptional regulators: structure 136 6.3 Specificity and selectivity of eukaryotic transcription factors 140 6.4 Sp1 transcription factor 142 6.5 Heat shock transcription factor (HSF) 142 6.6 How do transcriptional activators work? 148 6.7 Transcription by RNA polymerase III 153 6.8 The establishment of repression 154 References 156 Contents vii 7 Chromatin and transcription 158 7.1 Local control of chromatin structure 158 7.2 The structural organization of chromatin 166 7.2.1 Chromosomal superstructure 167 7.3 Heterochromatin 171 7.4 The regulation of transcriptional competence 172 References 175 Index 176 Preface Our understanding of the mechanisms regulating gene expression, which determine the patterns of growth and development in all living organisms, ultimately involves the elucidation of the detailed and dy namic interactions of proteins with nucleic acids - both DNA and RNA. Until recently the commonly presented view of the DNA double helix as visualized on the covers of many textbooks and journals - was as a monotonous static straight rod incapable in its own right of directing the processes necessary for the conservation and selective reading of genetic information. This view, although perhaps extreme, was reinforced by the necessary linearity of genetic maps. The reality is that the biological functions of both DNA and RNA are dependent on complex, and sometimes transient, three-dimensional nucleoprotein structures in which genetically distant elements are brought into close spatial proximity. It is in such structures that the enzymatic manipulation of DNA in the essential biological processes as DNA replication, transcription and recombination are effected - the complexes are the mediators of the 'DNA transactions' of Hatch Echols. In these manipulations the DNA no longer acts as a passive partner to the proteins - rather its physicochemical properties determine the pre ferred direction of the manipulation, be it the bending of the double helical axis or the separation of two strands of the double helix. In this scenario the proteins in the complex both add precision and keep the DNA molecule under tight control in keeping with the philosophical tenet that biological systems leave nothing to chance. Similar principles of course apply also to RNA-protein complexes where, for example, the catalytic activity of particular RNA species is modulated by interaction with proteins. The aim of this book is to give an overview of the function of DNA-protein complexes particularly taking into account the role of the structural flexibility and heterogeneity of the DNA molecule itself. In a short description of this kind it is impossible to be comprehensive. Instead, I have tried to choose particular examples which illustrate the general principles involved. Some of the choices may well be eclectic but, I hope, otherwise informative. Inevitably there are parts of this book that will be rapidly overtaken by new discoveries - the pace of contemporary research is now such that there are always new and interesting results that would merit inclusion. However a line must be drawn and I hope that the principles, if not the details, will remain valid. For the errors that remain I take full responsibility. Finally I would like to thank all those who have provided the spur for the completion of this book. There are all my colleagues at LMB who have been a constant source of stimulation and a rein to the more way ward ideas. I am particularly grateful to Horace Drew, whose constant espousal of a DNA -centric view of the world constituted the essential genesis of this account. Thank you too to the always encouraging and incredibly patient staff of Chapman & Hall - it has been a real pleasure to work with them. Lastly, I am especially indebted to my wife, who has constantly encouraged this enterprise and without whose support it would have assuredly never been finished. It is to her that this book is dedicated. Andrew Travers Cambridge 1 DNA structure 1.1 STRUCTURAL FEATURES OF DNA The initial step in the expression of any gene is the selection of that gene from among the many thousands encoded in a typical DNA genome. This selection invariably requires the interaction of protein molecules with specific sequences or structures in the DNA itself. To understand the physical and chemical basis for these interactions we must first con sider the structure of DNA and the ways in which this structure can change. The classical view of the structure of DNA molecules is derived largely from the X-ray analysis of oriented DNA fibres. In such fibres the pre ferred configuration consists of two antiparallel sugar-phosphate back bones wrapped in a right-handed double helix. The attached bases on one strand are directed approximately towards the axis of this double helix and form hydrogen bonds with their complementary bases on the other. On the exterior surface the sugar-phosphate backbones are separ ated successively by two grooves, termed the 'major' and 'minor' grooves. These grooves are defined structurally by the orientation of the base pairs (Figure 1.1) such that the N7 atom of the purine ring and the C5 atom of the pyrimidine ring face out into the major groove. It is these exposed atoms, and any attached chemical groups, which are directly accessible to interaction with external reagents, such as proteins, certain drugs, or reactive small molecules. A second aspect of DNA structure is the relationship of one base pair to its neighbours. For this purpose we can simplify the structure and consider each base pair as a planar domino (Figure 1.2), although in actuality the two bases in an individual base pair are usually twisted relative to each other about the long axis of the base pair ('propeller twist'). The degree of propeller twist is an important determinant of the external character of the double helix. The helical arrangement requires that each base pair be rotated relative to its immediate neighbour. This 2 DNA structure Major groove H \ N N-H······ '··0 /~N-H-N A-T base pair Deoxyribose N / o Deoxyribose Minor groove Major groove /Lj.):=~:~H=:~ < G--C base pair Deoxyribose N )-N\ N-H·················O Deoxyribose H Minor groove Figure 1.1 Geometry of A-T and G-C base pairs and their orientation relative to the major and minor grooves of DNA. rotation is measured with respect to the local long axis of the double helix and is termed 'twist'. This twist is variable, but for most right handed double helices it falls in the range of 22-45° between each successive base pair. A second important parameter is the angle between the planes of successive base pairs. The major observed departure from planar stacking is a rotation, termed 'roll', about the long axis of the base pairs. A rotation of this type results in a change of direction of the helical axis and thus can be directly related to the bending of a DNA molecule. A similar departure from planar stacking by a relative rotation about the short axis of the base pair, term 'tilt', is in general much smaller than roll.