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, currently supported by the NSF, 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 β-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 have determined its structure by NMR. Current work is directed towards determining the interface between the dimerization domain and the DNA binding domain in the presence and in the absence of arabinose. Genetic, biochemical, and computational methods are being used for this. Current work is also directed towards understanding what controls formation of the alternative structures the chameleon-like N-terminal arm of AraC.
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. Most recently we developed 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 araI1 half-sites and 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
Practical Methods in Molecular Biology, Robert Schleif,
Pieter Wensink, 1981, Springer-Verlag, New York. A
cookbook of practical knowledge and methods used in
Genetics and Molecular Biology 2nd Ed. Robert Schleif, 1993, Johns Hopkins Press. View or download the entire book in pdf format. (698 pages, 5.6 MB, Bookmarked). Purchase hardcopy. A graduate level textbook providing a rigorous and thoughtful presentation of the fundamentals of molecular biology.
Analysis of Protein Structure and Function: A Beginner's
Guide to CHARMM View or download
the entire book in pdf format (172 pages, 800 KB,
Bookmarked). Describes the operation and use of CHARMM for
molecular mechanics and molecular dynamics analysis of
protein coordinates, energetics, and motions.
Two Reviews of the Arabinose System
Other Recent Papers
Computational Predictions of the Mutant Behavior of AraC Monica Berrondo, Jeffrey J. Gray, and Robert Schleif, J. Mol. Biol. 398, 462-470 (2010). PMID: 20338183.
Active Role of the Interdomain Linker of AraC, Jennifer Seedorff and Robert Schleif, J. Bacteriol. 193, 5737-5746 (2011). PMID: 21840981
that Two Arabinose Molecules are Required for the Normal
Arabinose Response of AraC, Biochemistry 51, 8085-8091
(2012). PMID: 23002904
Understanding the basis of a class of paradoxical mutations in AraC through simulations, A. Damjanovic, B.T. Miller, and R. Schleif, Proteins 81, 490-498 (2013). PMID: 23150197
Lab and Teaching
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
Ph.D. Students Trained
Some Comments for Graduate Students
Advanced Molecular Biology Homepage
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?
Gamma, Perception, Posterization, and Raw Conversion
Sensing Violet: The Human Eye and Digital Cameras
Shortcuts and Useful Techniques for Picture Window Pro, an image editing program.
My Best from 2011 (17 images)
Best of 2012 (19 images)
Best of 2013 (11 images)
Random, Tennis and Science
Outcomes Related to Probability of Winning Individual
Optimum Poaching Strategy in Tennis Doubles
Affinity of Transcription Factor-RNAP Interactions
A Very Simple Derivation of the Boltzmann Distribution
Ligand binding to an homo-oligomeric protein, cooperativity, macro and micro dissociation constants
Dimer Binding Affinity in Terms of Monomer Binding Affinity