W. Michael Holmes , Ph.D.
Professor

Phone: (804) 828-2327
Dept. Fax: (804) 828-9946
e-mail: holmes@vcu.edu

Address:
Department of Microbiology & Immunology
Virginia Commonwealth University
PO Box 980678
Va Biotech Research Park #1, Suite 2-227 Lab 8
Richmond, VA 23298-0678

Professional Experience

• B.S., 1964, University of Memphis 
• M.S., 1968, University of Memphis 
• Ph.D., 1974, University of Tennessee School of Medicine 
• Postdoctoral, 1974-1977, University of California, Irvine
• "Chercher Associe", 1995, Institute de Biologie Moleculaire et Cellulaire, C.N.R.S. Strasbourg, France.
• 2005, Visiting Professor, UC Berkeley, Research with Dr. Jennifer Doudna.

Research Interests:

RNA protein interactions, Site specific RNA modification,  Micro RNA metabolism in mammalian cells; role in Cancer and and gene expression 

RNA modification and mechanisms of site specific RNA methylation.

Cellular RNA’s have now been shown to contain over 100 different modified nucleotides. These bases can be found in many domains of all types of cellular RNA to include tRNA in which over 80 modified bases can be found. It is notable that over one percent of the typical bacteria genome is dedicated to modifying RNA, and that these modifications are conserved across different species, suggesting that they must play an important role in cellular viability.    All cellular forms contain enzymes(s) that catalyze the S-Adenosyl-Methionine (AdoMet) dependent methylation of tRNA molecules at G37, a modification that, at least in bacteria and yeast prevent frameshifting. In eubacterial TrmD, the product of the trmD gene, carries out this reaction.  It appears that the enzyme acts through general base catalysis by deprotonating the N1 group of quanosine and allowing it to nucleophilically attack the methyl group of AdoMet. We have shown that only a subset of tRNA species containing G or A at position 36  and G at 37 are methylated by the E. coli trmD enzyme. Thus, other that histidine tRNA, only tRNA’s recognizing codons beginning with C are methylated by trmD. We also have shown that the entire tRNA structure is required for maximal catalytic activity, and we have more precisely identified tRNA sequences which make contact with the trmD enzyme during catalysis.

Recently, we determined the crystal structure of the E. coli trmD enzyme (Figure 1).  The enzyme is a homodimer of 30,000 molecular weight subunits. The binding site for AdoMet is constructed from different domains from both subunits and is deeply buried in the molecule.  We have shown for the first time that this protein contains a knotted peptide which is an intregral part of the AdoMet binding site (Figure 2).  More recently, we have found that the G36pG37 bases are flipped into the catalytic center of the enzyme prior to methylation. In addition it appears that the enzyme also undergoes substantial isomerization to carry out this interesting reaction.  We currently are studying the dynamic of this reaction using a variety of methods to include FRET (Fluorescence Energy Transfer) NMR, and various stop flow techniques. In addition, we are determining the structure of a number of interesting structural variants which may reveal the structural pathway to catalysis.  We are also interested in the identification of important inhibitors of trmD which may serve as a new class of antimicrobial agents. To this end we have also determined the structure of the Staphlococcus aureus trmD protein.

   Recently, we isolated the human gene which carries out G37 tRNA methylation which has been designated the Trm5 gene. We have found it is fundamentally different in structure and sequences required for tRNA recognition.  This is important if we are to identify inhibitors which do not inhibit the human enzyme.

We currently, are attempting to determine the crystal structure of this and another G37 methyltransferase from the Archeal organism Pyrococcus abbyssi. We believe that these enzymes have evolved quite separately from eubacteria enzymes. This information will allow us to study how these important structures have come about through cellular evolution.  

The cellular modifisome

   We have recently found that the human Trm5 enzyme is found in complex with other cellular modification enzymes. In addition, we believe that this enzyme can be localized to the mitochondia, and be imported back into the nucleus where it can complex with other modification enzymes. The large 3’ UTR contains a HMG17 open reading frame which may be involved in the transport back into the nucleus where we propose it might associate with transcribing tRNA genes. This would provide an excellent vectorial mechanism for modifying tRNA as it is transcribed.  We are interested in examining all the possible steps in this import pathway of RNA metabolism. 

The function of thermophilic trmD proteins

  We have isolated and are studying the trmD protein  form Thermotoga maritima. This interesting enzyme must function in vivo at high temperatures since the organism from which it is derived grows optimally at 90C. Therefore, we wish to understand how the protein and the tRNA it must methylate remain stable at such high temperatures. We have found that this enzyme actually is inactive at ambient temperatures; therefore, heat must somehow activate the protein.  At 90 degrees tRNA is well know to be completely denatured. It appears that the interaction of tRNA with the trmD enzyme and the important factor eFTu must explain this remarkable stability.  

Human mRNA metabolism, localization and the role of micoRNA metabolism in gene expression and neoplasia

  We have been studying the role of 3' untranslated sequences in selected human mRNA which may serve as "Zip codes" in the cell for placing mRNA in the correct place in the cytoplasm. Messages we have selected for study encode for various cytoskeletal and contractile elements such as vimentin and certain forms of actin. We are characterizing specific proteins which interact with these mRNA species and ultimately wish to understand the cellular machine which localizes these important mRNA species. One protein we have isolated has a remarkable affinity for a specific mRNA structure and can only be removed by RNAase. We currently believe this protein is part of the machine which serves to localize vimentin mRNA and perhaps participates in the site specific translation of mRNA. We believe that such 3' UTR transduction sequences may also serve as cellular signals which communicate with major signal transduction pathways in the cell. Thus, RNA can now be viewed as a important device for intercellular communication. Finally, we are determining if the vimentin mRNA binding sequence can be a target for molecules and drugs which can selectively shut down mRNA expression. This may become another key target for drug development. Recently, it has been shown that the expression of the vimentin gene is essential if prostate cancer cells are to become metastatic.  It now appears that the 3’ UTR of the vimentin mRNA interacts with at least two microRNA species. One, appears to interact at the site where the HAX localization protein binds. We believe it is possible therefore that microRNA species may be involved in mechanisms of mRNA localization.  MicroRNA’s are emerging as important potential targets for therapy.  We are extending these studies to other genes important for the metastatic phenotype  and are developing the means for microarray analysis for all know cellular micro RNA species in selected human tumor cells.

Selected Publications:

Wahab, S.Z., W.M. Holmes and Z.E. Zehner. Both 5' and 3'flanking sequences are required for the expression of a human tRNAmeti gene. Gene 77:361-371 (1989).

Robefts, I., P.B. Hylemon and W.M. Holmes. Rapid method for altering Bacterial Ribosomebinding sequences for over expression of proteins in Escherichia coli. Protein Exp. and Purification 2:117-121 (1991).

Holmes, W.M., I.R. Roberts, C. Andraos-Selim and S.Z. Wahab. RNA Structures required for TRNA methylation: E coli TRNA, le, methylation by homologous 1 -methyl Guanosine Transferase. J. Biol. Chem. 267:13440-134 (1992).

Bauer, B.F., R. Elford, and W.M. Holmes. Mutagenesis and functional analysis of the Escherichia coli tRNAl Leu promoter. Molecular Microbiol. 7:265-273 (1993).

Wahab, S.,Rowley, K. and W.M. Holmes. Effects of tRNAl Leu over-production in Escherichia coli. Molecular Microbiology. 7:253-263.

Rowley, K.B., , R.M. Elford, I.R. Roberts, and W.M. Holmes. In vivo regulatory responses of four Escherichia coli operons which encode leucyltRNAs. J. Bacteriol. 175:1309-1315 (1993).

Holmes, W. M., C. Andraos-Selim, and M. Redlak. 1995. tRNA-M1G methyltransferase interactions: touching bases with structure. Biochimie. 77:62-65.

Zehner, Z. E., R. K. Shepherd, J. Gabryzuk, M. Al-Ali, and W. M. Holmes. 1997. Protein:RNA interactions within the 3' untranslated Region of Vimentin mRNA. In Press, Nucleic Acids Research.

Redlak, M., C. Andraos-Selim, R. Giege, C. Florentz, and W. M. Holmes. 1997.

Interaction of tRNA with tRNA (guanosine-1_ methyltransferase; binding specificity determinants involve the dinucleotide G36pG37 and tertiary structure. Biochemistry 36:8699-8709.

J. Gabryszuk, R. Tyler-Cross, and W. M. Holmes. Interaction of tRNA with a RNA modification enzyme:structural changes accompany binding and recognition. Nucleic Acids Symp Ser. 1997;(36):104-6.

Shepherd RK, Gabryszuk J, Al-Ali M, Allen CA, Joyce I, Holmes WM, Zehner ZE. A dual stem-loop structure in the 3'untranslated region of vimentin mRNA binds specific protein. Nucleic Acids Symp Ser. 1997;(36):142-5.

Brule H, Holmes WM, Keith G, Giege R, Florentz C. Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern. Nucleic Acids Res. 1998 Jan 15;26(2):537-43.

Pokholok, D.K., Redlak, M., Turnbough, C.L. Jr., Dylla, S., and W. Michael
Holmes. 1999. Multiple Mechanism Are Used for Growth Rate and Stringent Control of leuV Transcriptional Initiation in Escherichia coli. J. Bacteriol. 181: 5771-5782.

Perreau, V, Keith, G., W. M. Holmes, Przykzorska, A., Santos, M. A.S., and Tuite, M. F. The Candida albicans CUG-decoding ser-tRNA has an atypical anticodon stem-loop structure.1999.  J. Mol. Biol. 293:1039-1053.

Vulgaris, J. Pokholok, D., Holmes, W. M., and C. Squires. 2000. The Feedback Response of E.coli rRNA Synthesis is not Identical to the Mechanism of Growth Rate-Dependent Control.  J.Bacteriol. 182:536-539.

Al-Maghrebi, M., Brule, H., Padkina, M., Allen, C., Holmes, W.M., and Zehner, Z.E. 2002. The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1. Nucleic Acids Res. 2002 Dec 1;30(23):5017-28.

Elkins, P.A., Watts, J.P., Zalacain, M., van Thiel, A., Vitazka, P. R., Redlak, M., Andraos-Selim, C., Rastinejad, F., and W. M. Holmes. 2003. Insights into catalysis by a knotted TrmD tRNA methyltransferase. J. Mol. Biol. 333/5: 931-949.

O’Dwyer, Watts, J.M., Biswas, S., Ambrad, J., Barber, M., Brule, H., Petit, C., Holmes, D.J., Zalacain, M., W.M. Holmes. 2004. Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for Growth. J. Bacteriol. 186:2346-2354.

Opel, M.L., Aeling, K., Holmes, W.M., Johnson, R., Benham, C., and G. W. Hatfield. 2004. Activation of Transcription Initiation from a Stable RNA Promoter by a Fis Protein-mediated DNA Structural Transmission Mechanism. Mol. Microbiol.  Jul;53(2):665-74.

Brule, H., Elliott, M., Redlak, M., Zehner, Z., and W.M. Holmes. 2004. Isolation and Characterization of the Human tRNA-(N1G37) Methyltransferase (TRM5) and comparison to the E. coli TrmD protein. Biochemistry. 2004 Jul 20;43(28):9243-55.

Watts, J. M.,  Gabruzsk, J., and Walter M. Holmes. 2005. Ligand-Mediated Anticodon conformational Changes Occur during tRNA Methylation by a TrmD Methyltransferase. Biochemistry. 44:”6629-6639.

Watts, J. M. and Walter M. Holmes. 2006. Mechanisms of tRNA methylation by the Thermotoga maritima TrmD protein. In preparation. For submission to Biochemistry.

Watts, J. M. and Walter M. Holmes. 2006.  Structural requirements for AdoMet binding by the E. coli TrmD protein. In preparation for submission to Nuc. Acids Research

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Date Last Modified: 07/10/2008
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