B.S., University of Delaware, 1990, Honors
PhD, University of Pennsylvania, 1996, (Barry S. Cooperman)
Postdoctoral Research Associate 1996-1997, MIT, (Paul Schimmel)
Postdoctoral Research Associate 1997-1999, The Scripps Research Institute, (Paul Schimmel)
Office: Salem 108
Phone: (336) 758-5568
Home Page: http://www.wfu.edu/~alexanr/Alexander_Lab2.htm
Understanding protein-nucleic acid interactions at the molecular level
Our research program is primarily centered on understanding the mechanisms of protein synthesis. Translation of a single protein from its nucleic acid precursor requires dozens of cellular components. Amino acids are assembled into polypeptides at the ribosome, a large ribonucleoprotein complex where the genetic message is decoded. Individual proteins play essential roles in maintaining the accuracy of translation. The aminoacyl-tRNA synthetases attach amino acids to transfer RNA (tRNA) molecules, thereby establishing the genetic code that dictates which amino acid matches which trinucleotide codon. Other protein factors facilitate the three steps of translation: initiation, elongation, and termination. For example, factors recognize initiation and termination signals, help assemble functional complexes, recruit aminoacyl-tRNA molecules, and trigger release of the full-length protein.
Although the basic mechanisms of protein synthesis are established and structures of many of the components have been determined, details remain unknown at the molecular level. Not only are the mechanisms of protein synthesis worth investigating at the level of basic research, but they provide many targets for design and development of new drugs. Because translation is an essential function in all organisms, inhibitors of protein synthesis are among the most common drugs in use today. The projects in our lab use a variety of techniques (protein engineering, kinetic analysis, binding studies, PCR amplification, nucleic acid synthesis and purification, and spectroscopy) to answer biochemical questions on a molecular level. We also have collaborations with researchers who use X-ray crystallography, mass spectrometry, and bacterial and mammalian cellular analyses to probe molecular mechanisms.
Contribution of conformational flexibility to chemical catalysis
In addition to being key players in translation, the aminoacyl-tRNA synthetases (aaRSs) are good models for understanding signal transduction-like events. Many aaRSs bind to the anticodon portion of their matching (cognate) tRNA molecules, and the anticodon-binding site is often some distance removed from the enzyme active site, where amino acid attachment occurs. Efficient catalysis therefore depends on communication between protein domains. Our lab investigates the contributors to domain-domain communication in methionyl-tRNA synthetase (MetRS), an aaRS that requires anticodon binding for efficient catalysis yet also aminoacylates a small tRNA mimic lacking an anticodon.
A comparison of the crystal structure of MetRS (shown here) with glutaminyl-tRNA synthetase (GlnRS) identified a similar peptide motif at the interface of catalytic and anticodon-binding domains in each protein. As this peptide is at the physical interface between functional domains, and as we expect conformational flexibility is important for catalysis, we introduced cysteine residues at positions that we thought would generate a disulfide bond to limit flexibility of this peptide. Functional analysis showed that this engineered disulfide dramatically reduced tRNA aminoacylation without affecting function of the individual domains. We are excited to have cut the functional communication through a single covalent bond, and will continue to characterize this and other mutants using kinetic assays, spectroscopy, and crystallography. We are also making specific nucleotide substitutions in tRNA Met to correlate sequence and structure to inter-domain communication.
The side-effects of accuracy control
The aminoacyl-tRNA synthetases are responsible for maintaining the fidelity of the genetic code by accurately attaching amino acids with their matching tRNAs. In some cases, synthetases actively prevent mis-incorporation of amino acids into proteins by hydrolyzing noncognate aminoacyl adenylates or aminoacyl-tRNAs. One of the amino acids that may be processed this way by MetRS is homocysteine, which lacks the terminal methyl group of methionine. For some time there has been clinical evidence that high serum homocysteine levels are correlated with diseases such as arthrosclerosis and lupus. We are interested in investigating the link between MetRS and the chemical intermediates formed when homocysteine is edited out of the biosynthetic pathway. We are working with investigators at the Scripps Research Institute in California to study the role of MetRS in human disease.
The impact of minor-groove DNA adducts on cellular processes
We have been collaborating with Dr. Uli Bierbach (WFU Chemistry) to study the molecular effects of treating DNA with the novel platinum-containing compound PT-ACRAMTU. We have confirmed using restriction enzyme analyses and transcriptional footprinting that PT-ACRAMTU modifies A bases in TpA and GpA steps. Covalent modification inhibits T7 RNA polymerase from transcribing DNA, which may point to a mechanism for its demonstrated activity against cancer cells. Electrophoretic mobility shift assays with the TATA-binding protein (TBP) demonstrated that adducts formed at the N3 position of adenine are most effective at blocking TBP binding and probably represent the physiologically relevant adduct. We will continue to probe the molecular impact of DNA damage using the tools of biochemistry and molecular biology.
Characterizing a cold-induced RNA helicase
We are working with Dr. Pamela Jones at Winston-Salem State University to characterize the activity of CsdA, a cold-induced protein found in bacteria. CsdA has all the canonical sequence motifs attributed to the DEAD-box helicases, which are ATP-dependent RNA helicases, or enzymes that unwind RNA. We have determined that CsdA indeed unwinds certain RNA substrates in the presence of ATP. The DEAD (Asp-Glu-Ala-Asp) sequence of CsdA is essential for this function. These results parallel those observed in vivo by Dr. Jones. We are investigating the substrate specificity for this helicase.
M.E. Budiman, U. Bierbach, and R.W. Alexander (2005) “DNA minor groove adducts formed by a platinum-acridine conjugate inhibit association of TATA-binding protein with its cognate sequence.” Biochemistry44, 11262-11268.
M.E. Budiman, R.W. Alexander, and U. Bierbach (2004) “Unique base-step recognition by a platinum-acridinylthiourea conjugate leads to a DNA damage profile complementary to that of the anticancer drug cisplatin.” Biochemistry43, 8560-8567.
R.W. Alexander, and K. Tamura (2004) “Peptide synthesis through evolution.” Cell. Mol. Life Sci.61, 1317-1330.
R.W. Alexander, and P. Schimmel (2001) “Domain-domain communication in aminoacyl-tRNA synthetases.” Prog. Nucl. Acid Res. Mol. Biol. 69, 317-349.
R.W. Alexander and P. Schimmel (2002) “Protein synthesis.” In Encyclopedia of Physical Science and Technology (Robert A. Myers, ed.) 3 rd ed., Vol. 13. Academic Press ( San Diego, CA), 219-240.
R.W. Alexander and P. Schimmel (2000) “Multifunctional proteins.” In McGraw-Hill 2001 Yearbook of Science & Technology, McGraw-Hill ( New York, NY), pp.265-266.
B. S. Cooperman, R. W. Alexander, Y. Bukhtiyarov, S. N. Vladimirov, Z. Druzina, R. Wang, and N. Zuno (2000) “Photolabile derivatives of oligonucleotides (PHONTs) as probes of ribosomal structure.” Methods Enzymol. 318, 118-136.
R.W. Alexander and P. Schimmel (1999) “Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex.” Biochemistry38, 16359-16365.
R. Wang, R.W. Alexander, M. van Loock, S. Vladimirov, Y. Bukhtiyarov, S.C. Harvey, and B.S. Cooperman (1999) “Three-dimensional placement of the conserved 530 loop of 16 S rRNA and of its neighboring components in the 30 S subunit.” J. Mol. Biol. 286, 521-540.
R.W. Alexander, B.E. Nordin, and P. Schimmel (1998) “Activation of microhelix charging by localized helix destabilization.” Proc. Natl. Acad. Sci. USA95, 12214-12219.
P. Schimmel and R.W. Alexander (1998) “Diverse RNA substrates for aminoacylation: clues to origins?” Proc. Natl. Acad. Sci. USA 95, 10351-10353.
P. Schimmel and R.W. Alexander (1998) “All you need is RNA.” Science 281, 658-659.