Search

Using a novel approach that combines high temporal resolution of the laser T-jump technique with unique sets of fluorescent probes, this study unveils previously unresolved DNA dynamics during search and recognition by an architectural DNA bending protein and two DNA damage recognition proteins. Many cellular processes involve special proteins that bind to specific DNA sites with high affinity. How these proteins recognize their sites while rapidly searching amidst ~3 billion nonspecific sites in genomic DNA remains an outstanding puzzle. Structural studies show that proteins severely deform DNA at specific sites and indicate that DNA deformability is a key factor in site-specific recognition. However, the dynamics of DNA deformations have been difficult to capture, thus obscuring our understanding of recognition mechanisms. The experiments presented in this thesis uncover, for the first time, rapid (~100-500 microseconds) DNA unwinding/bending attributed to nonspecific interrogation, prior to slower (~5-50 milliseconds) DNA kinking/bending/nucleotide-flipping during recognition. These results help illuminate how a searching protein interrogates DNA deformability and eventually “stumbles” upon its target site. Submillisecond interrogation may promote preferential stalling of the rapidly scanning protein at cognate sites, thus enabling site-recognition. Such multi-step search-interrogation-recognition processes through dynamic conformational changes may well be common to the recognition mechanisms for diverse DNA-binding proteins.

The structural biology of protein-nucleic acid interactions is in some ways a mature field and in others in its infancy. High-resolution structures of protein-DNA complexes have been studied since the mid 1980s and a vast array of such structures has now been determined, but surprising and novel structures still appear quite frequently. High-resolution structures of protein-RNA complexes were relatively rare until the last decade. Propelled by advances in technology as well as the realization of RNA's importance to biology, the number of example structures has ballooned in recent years. New insights are now being gained from comparative studies only recently made possible due to the size of the database, as well as from careful biochemical and biophysical studies. As a result of the explosion of research in this area, it is no longer possible to write a comprehensive review. Instead, current review articles tend to focus on particular subtopics of interest. This makes it difficult for newcomers to the field to attain a solid understanding of the basics. One goal of this book is therefore to provide in-depth discussions of the fundamental principles of protein-nucleic acid interactions as well as to illustrate those fundamentals with up-to-date and fascinating examples for those who already possess some familiarity with the field. The book also aims to bridge the gap between the DNA- and the RNA- views of nucleic acid - protein recognition, which are often treated as separate fields. However, this is a false dichotomy because protein - DNA and protein - RNA interactions share many general principles. This book therefore includes relevant examples from both sides, and frames discussions of the fundamentals in terms that are relevant to both. The monograph approaches the study of protein-nucleic acid interactions in two distinctive ways. First, DNA-protein and RNA-protein interactions are presented together. Second, the first half of the book develops the principles of protein-nucleic acid recognition, whereas the second half applies these to more specialized topics. Both halves are illustrated with important real life examples. The first half of the book develops fundamental principles necessary to understand function. An introductory chapter by the editors reviews the basics of nucleic acid structure. Jen-Jacobsen and Jacobsen discuss how solvent interactions play an important role in recognition, illustrated with extensive thermodynamic data on restriction enzymes. Marmorstein and Hong introduce the zoology of the DNA binding domains found in transcription factors, and describe the combinational recognition strategies used by many multiprotein eukaryotic complexes. Two chapters discuss indirect readout of DNA sequence in detail: Berman and Lawson explain the basic principles and illustrate them with in-depth studies of CAP, while in their chapter on DNA bending and compaction Johnson, Stella and Heiss highlight the intrinsic connections between DNA bending and indirect readout. Horvath lays out the fundamentals of protein recognition of single stranded DNA and single stranded RNA, and describes how they apply in a detailed analysis of telomere end binding proteins. Nucleic acids adopt more complex structures - Lilley describes the conformational properties of helical junctions, and how proteins recognize and cleave them. Because RNA readily folds due to the stabilizing role of its 2'-hydroxyl groups, Li discusses how proteins recognize different RNA folds, which include duplex RNA. With the fundamentals laid out, discussion turns to more specialized examples taken from important aspects of nucleic acid metabolism. Schroeder discusses how proteins chaperone RNA by rearranging its structure into a functional form. Berger and Dong discuss how topoisomerases alter the topology of DNA and relieve the superhelical tension introduced by other processes such as replication and transcription. Dyda and Hickman show how DNA transposes

This volume represents the first collection of articles contributed by research leaders working on the Myb family of transcriptional regulatory proteins. In more than twenty chapters the authors discuss the range of biological processes and diverse cell types in which Myb proteins operate. Although concentrating on the three vertebrate Myb family members, homologues from lower species are also discussed because of the light they are able to shed on the evolution and function of these proteins. Individual chapters describe the involvement of Myb proteins, in particular c-Myb, in normal and diseased development and function of many tissues including haemopoietic cells, blood vessels, the gastrointestinal tract and the brain. Several chapters explore the mechanistic details of the action of Myb proteins, especially structural features, their interaction with DNA and other regulatory proteins, and the variety of genes that are regulatory targets for this group of transcription factors. This work will be of interest to those working directly in the field and also to the wider research community investigating the transcriptional regulation of development, differentiation and growth. The therapeutic potential of manipulating Myb function is also discussed making the book appealing to clinician scientists in several fields including haematology, oncology and cardiology.

Footprinting of Nucleic Acid-Protein Complexes provides protocols for studying the stoichiometry, binding site size and location, and structural changes in nucleic acids caused by their interaction with proteins. The methods are central to studying key biological processes, such as transcription and translation. The techniques are important to experiments in vivoand in vitro, in eukaryotes and in prokaryotes, at qualitative and quantitative levels,and across many disciplines.This book is a laboratory manual of footprinting techniques for studying nucleic acid-protein interactions. It contains clear and concise descriptions of the most important methodologies, and includes in vivo as well as in vitro applications. It is aimed at bench scientists from graduate students on, and should be of value in industrial labs as well as in academic settings. Use of different footprinting approaches can provide unique insights into DNA-protein systems. The protocols containedin this handbook are written to be'user-friendly,'and thus should be conducive to extending the use of footprinting to new systems. The section on quantitative analysis of DNAse I footprints should prove especially useful for in depth evaluation of cooperative interactions.(For the End User)Provides clear exposition of footprinting techniques for characterizing DNA-protein interactionsCovers both protection methods for identifying sites of protein binding and interference methods for determining points of contact between DNA and proteinIncludes approaches for both in vitro and in vivo measurementsHigh quality, timely, and of lasting practical value in the laboratory