Rebecca Alexander Assistant Professor 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)

Mailing Address: Chemistry Department, Wake Forest University, Winston-Salem, NC 27109.
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Our research program is 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 three projects described below are aimed at understanding critical components of the protein synthesis machinery.  These projects use a variety of techniques (protein engineering, kinetic analysis, binding studies, PCR amplification, nucleic acid synthesis and purification) to answer biochemical questions on a molecular level.

Contributions to long-range signaling in an aminoacyl-tRNA synthetase

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 will investigate 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.  This study will have a two-pronged approach: site-directed mutagenesis of MetRS and selection of minimal substrates of tRNAMet.

Project 1.  Structural contributions to signaling in MetRS

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.  In order to determine whether the structure of this motif contributes to communication between the domains, we will use cassette mutagenesis to construct chimeric proteins containing the linker peptide of GlnRS within the context of MetRS (and vice versa).  These chimeric proteins will be evaluated for tRNA binding and aminoacylation functions.  A randomized protein pool will also be generated such that each position of the interface peptide has the amino acid residue of either MetRS or GlnRS.  A genetic screen will identify proteins from this population able to efficiently aminoacylate tRNAMet.  Such a thorough investigation of sequence possibilities should provide clues as to which residues or sequences contribute to communication between the domains of MetRS.

Project 2.  Selection of minimal efficient RNA substrates of MetRS

A microhelix mimic of the tRNAMet acceptor stem is sequence-specifically aminoacylated by MetRS, although the catalytic efficiency of the reaction is significantly decreased.  Both the microhelixMet and an anticodon stem-loop mimic bind to MetRS, but the presence of the anticodon stem-loop does not increase the rate of microhelix aminoacylation.  At a minimum, therefore, the two portions of tRNAMet must be covalently linked.  In an attempt to re-establish communication between the anticodon and acceptor stem portions of tRNAMet, we will connect the RNA microhelices with a flexible linker of varying length and evaluate the aminoacylation kinetics.  Once the correct substrate orientation on MetRS is established, we will insert randomized nucleotide linkers and identify improved RNA substrates by in vitro selection.  It is anticipated that such a selection will provide clues as to which regions of MetRS are involved in long-range signaling between anticodon binding and active site domains.

Project 3.  Bacterial initiation factor 2: a possible drug target?

Initiation of protein synthesis is quite different in bacteria and eukaryotes.  This makes the protein initiation factors (IFs) good targets for potential new drugs, if we can identify inhibitors of bacterial IFs that have no effect on human IFs.  Bacterial IF-1 is a small protein (72 amino acids in E. coli) whose structure has been solved by NMR although its precise function is not known.  It has been predicted that IF-1 is an RNA-binding protein but this has not yet been demonstrated.  We will overexpress IF-1 and generate photoreactive variants suitable for crosslinking studies.  Previous studies showed that IF-1 binds to the small ribosomal subunit and can be crosslinked to ribosomal proteins.  We will generate crosslinks from modified IF-1 to components of 30S initiation complex, which includes mRNA, initiator tRNA, and the small (30S) ribosomal subunit.  We anticipate this will produce a model of IF-1 binding within the initiation complex, which may shed light on its function.  If, as anticipated, IF-1 binds to one or more RNAs in the complex, we will begin an in vitro selection protocol to identify nuclease-resistant inhibitors of bacterial IF-1 using a mirror-image approach.

Recent Publications

R.W. Alexander and P. Schimmel "Protein synthesis." In Encyclopedia of Physical Science and Technology (Robert A. Myers, ed.)  3^rd ed. Academic Press, San Diego.  In press.

R.W. Alexander and P. Schimmel  "Multifunctional proteins." In McGraw-Hill 2001 Yearbook of Science & Technology, McGraw-Hill, New York. In press.

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." Biochemistry 38, 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. USA 95, 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.

R.W. Alexander, and B.S. Cooperman (1998) "Ribosomal proteins neighboring 23 S rRNA nucleotides 803-811 within the 50 S subunit." Biochemistry 37, 1714-1721.

P. Muralikrishna, R.W. Alexander, and B.S. Cooperman (1997) "Placement of the a-sarcin loop within the 50 S subunit: Evidence derived using a photolabile oligodeoxynucleotide probe."  Nucleic Acids Res. 25, 4562-4569.

B.S. Cooperman, R.W. Alexander, and P. Muralikrishna (1995) "Photolabile oligoDNA probes of Escherichia coli internal ribosomal structure."  Nucleic Acids Symposium Series 33, 59-62.

R.W. Alexander, P. Muralikrishna, and B.S. Cooperman (1994) "Ribosomal components neighboring the conserved 518-533 loop of 16S rRNA in 30S subunits."  Biochemistry 33, 12109-12118.

K.V. Rosen, R.W. Alexander, J. Wower, and R.A. Zimmermann (1993) "Mapping the central fold of transfer RNA₂ ^fMet in the P-site of the Escherichia coli ribosome." Biochemistry 32, 12802-12811.

B.S. Cooperman, P. Muralikrishna, and R.W. Alexander (1993) "Photolabile oligodeoxyribonucleotide probes of E. coli ribosome structure."  In The Translational Apparatus, Knud H. Nierhaus, et al., eds. Plenum Press, New York, pp. 465-476.