239 Mudd Hall
Department of Biology
Johns Hopkins University
3400 N. Charles Street
Baltimore, MD 21218-2685
Office 410 516-5206
Lab 410 516-5207
Departmental fax 410 516-5213
Ph.D.University of California, Berkeley
Research in my laboratory is directed at understanding the basic biochemical and biophysical principles involved in protein function through the combined use of biochemistry, genetics, genetic engineering, and biophysics. Our criterion for understanding is that we can design and build systems that actually work and make use of these principles. Since we have had extensive experience with the arabinose operon and systems related to it and we have a large collection of mutations in AraC and the regulatory region as well as many mutant DNA's and proteins, many of our ongoing studies use this system. The ara system permits economic and rapid handling of the biology while displaying most of the repertoire of protein-protein, protein-DNA and gene regulatory principles that are found in prokaryotes and eukaryotes.
In 1984 we made the original discovery of DNA looping, a mechanism now known to be widely used in biology. More recently we discovered the two domain structure to AraC and grew the crystals from which the structure of the dimerization domain was determined. This work in connection with biochemical and genetic studies led us to the discovery of the role of the N-terminal arms on AraC and the "light switch" mechanism by which the arms of the protein regulate the looping-unlooping activity of the protein. The light switch mechanism and the use of arms in domain-domain and protein-protein interactions may be widespread in nature, and we are examinining its occurrence. Recently we demonstrated that the light switch mechanism can be ported to other proteins, and we have constructed a b-galactosidase whose activity is controlled by the light switch mechanism from AraC. The enzyme's activity is modulated by the presence of arabinose. Additionally, we have constructed other and simpler "man-made" regulatory proteins.
Several years ago we found that the DNA binding domain of AraC may readily be overproduced and purified. It appears to be a very good material for NMR studies, and we are now determining its structure by NMR as well as mapping its interactions with other proteins and with DNA. Current work is also directed towards improving our understanding of the electrostatics of protein-DNA interactions, what controls which of alternative structures the chameleon-like N-terminal arm of AraC assumes, the basic forms of allsteric regulation displayed by gene regulatory proteins, and the physical basis of altered properties of AraC mutants.
Approaches commonly used in the laboratory include biochemistry, genetics, genetic engineering, physiological
measurement, and biochemical and physical-chemical approaches, for example crystallography, fluorescence,
electrophoresis, plasmon resonance, NMR, as well as computational approaches. Our primary, but not only,
subject for comparison of theory and experiment is AraC protein.
Frequently we develop new experimental techniques to facilitate our studies. In the past we developed the DNA migration retardation assay so that biochemically meaningful information could be obtained from it and developed the missing contact method for determining specific amino acid-base interactions in DNA. More recently we developed methods for: locating linker regions in multi-domain proteins, constructing functional chimeric proteins when the domain locations are unknown, precise comparison of DNA binding affinities, and refolding DNA-binding proteins from insoluble inclusion bodies. We are now developing a method for investigation of the very weak protein-protein and domain-domain interactions that are often found in complex regulatory systems.
Summary of the Regulation Mechanism of the Arabinose Operon
The gene products of the arabinose operon in Escherichia coli enable the cells to take up and catabolize the five carbon sugar, L-arabinose. In the absence of arabinose, the dimeric AraC protein actively represses its own synthesis and the synthesis of the AraB, AraA, and AraD gene
products by binding to the araO2 and araI1half-sitesand forming a DNA loop that blocks access of RNA polymerase to the pC and pBAD promoters. Upon the addition of arabinose, AraC ceases looping and binds instead to the adjacent half-sites, araI1 and araI2, where it and the cyclic AMP binding protein, CAP, both help RNA polymerase to bind to the pBAD promoter and speed the formation of open complex by RNA polymerase, thereby stimulating the synthesis of the AraB, AraA, and AraD gene products 100- to 500-fold.
AraC protein consists of two loosely connected domains, a DNA-binding domain that both binds to the various I-like sites and which also interacts with RNA polymerase to activate transcription, and a dimerization domain that also binds arabinose. AraC protein is caused to form the DNA loop between the I1 and O2 half-sites by the N-terminal arms that extend from the dimerization domains and bind to the back of the DNA binding domains. The simultaneous interaction of these arms with both the dimerization domains and the DNA binding domains holds the DNA binding domains in a relative orientation that energetically favors DNA loop formation and disfavors binding to the direct repeat I1 and I2 half-sites. Upon the binding of arabinose to the dimerization domains, however, the N-terminal arms restructure such that the DNA binding domains are released and are thus freed to assume any relative orientation they like. As a result, they now prefer to bind to the two adjacent, half-sites I1 and I2, where such binding activates transcription from pBAD.
Books and Recent Publications
Genetics and Molecular Biology 2nd Ed.
View or download the entire book in pdf format.
A graduate level textbook providing a rigorous and thoughtful presentation of the fundamentals of molecular biology. Robert Schleif , © 1993 Johns Hopkins Press, Reproduced with Permission (698 pages, 5.6 MB, Bookmarked) Purchase hardcopy
Analysis of Protein Structure and Function: A Beginner's Guide to CHARMM View or download the entire book in pdf format. Describes the operation and use of CHARMM for molecular mechanics and molecular dynamics analysis of protein coordinates, energetics, and motions. (172 pages, 800 KB, Bookmarked)
Two Reviews of the Arabinose System
Other Recent Papers
The Salt Dependence of the Interferon Regulatory Factor 1 DNA Binding Domain Binding to DNA Reveals Ions Are Localized around Protein and DNA, V. V. Hargreaves and R. F. Schleif, Biochemistry 47, 4119-28 (2008).
Undergraduate Students:Sam Kim
Graduate Students:Jennifer Seedorff
Research Scientist:Michael Rodgers
Lab and Teaching
Laboratory Methods These describe in recipe format how to do many routine molecular biology and biochemistry procedures. View or download the set of 160 pages that print on 5" x 8" index cards. Ours are kept in a recipe box and each category is printed on card stock of a different color.
Photographic: A Few Tutorials and Some of My Better Pictures
The Principles Behind Digital Image Sharpening
The Resolution of Digital Cameras: How Much is Needed and How Much Have You Got?
Sensing Violet: The Human Eye and Digital Cameras
Shortcuts and Useful Techniques for Picture Window Pro, an image editing program.
The Blizzard of '03
Forest Pictures (4)
Cornwall England 2003
Nature Pictures from Biology Retreat, October 2004
Canyonlands of the American Southwest,2005
Switzerland 2006 (12pictures)
Madrid and Baeza, Spain 2006 (6 pictures)
San Francisco Bay Area March 2007 (10 pictures)
Mostly Flowers, 2007 (11 pictures)
Hopkins Campus, 2008 (7 pictures)
Decent Pictures 2008-2009 (10 pictures)
Fall and Winter 2009 (7 pictures)
From a Hiking Tour of New Zealand (37 pictures) (Best viewed by maximizing browser viewing area)
Random, Perhaps Some with Scientific Interest
Tennis Outcomes Related to Probability of
Winning Individual Points
Optimum Poaching Strategy in Tennis Doubles
Affinity of Transcription Factor-RNAP Interactions
A Monte Carlo Charmm script for positioning domains or proteins subject to long distance constraints.