William E. Stumph
Professor
Biochemistry
Office: CSL 334
Office Phone: 619-594-5575
e-mail: wstumph@sciences.sdsu.edu
Research Interests
Research in Dr. Stumph's lab is aimed at understanding the molecular mechanisms of gene expression in higher organisms. Dr. Stumph's group has been characterizing and studying the expression of genes that code for the small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6. The snRNAs are a special class of RNA molecules that are involved in messenger RNA splicing (the removal of intervening sequences from messenger RNA precursors). The genes coding for the U-snRNAs are interesting to study not only because they code for RNA molecules with such fundamental importance to cellular metabolism, but also because their expression appears to be controlled by unique mechanisms. For example, most snRNA genes are transcribed by RNA polymerase II (the same enzyme that synthesizes messenger RNAs), but these Pol II-transcribed snRNA genes contain no introns and they lack the TATA sequences commonly found upstream of mRNA transcription units. Surprisingly, and in contrast, U6 snRNA genes contain TATA sequences but are transcribed by RNA polymerase III.
Despite this difference in RNA polymerase specificity, the promoter sequences of U6 genes are actually very similar to the promoters of U1-U5 genes. A 21-base-pair-long cis-acting sequence, termed the proximal sequence element, or PSE, is uniquely required for the transcription of both classes of snRNA genes. This PSE is recognized sequence-specifically by a multi-subunit protein termed the PSE-binding protein, PBP. Our goal is to understand how the same protein, PBP, can in one case recruit RNA polymerase II but in the other case recruit RNA polymerase III. In order to answer this question, we use the fruit fly Drosophila melanogaster as a model system, and we employ various biochemical, genetic, and molecular biological techniques.
As mentioned above, the PSEs of the Drosophila U1 and U6 genes are each 21 base pairs in length, and they are both recognized and bound sequence-specifically by the Drosophila melanogaster PSE-binding protein, DmPBP. The U1 and U6 PSE sequences are identical at 16 of the 21 positions. However, we have recently shown that the 5 nucleotide differences between them are sufficient to determine the RNA polymerase specificity of the U1 or U6 gene promoter. Furthermore, by site-specific protein-DNA photocrosslinking techniques, we have determined that DmPBP consists of at least three distinct polypeptide chains that contact the DNA of the PSE. These three subunits are designated DmPBP45, DmPBP49, and DmPBP95 based upon their apparent molecular masses. Significantly, the data also reveal that the precise contacts made between the protein subunits and the DNA are different depending upon whether DmPBP is bound to a U1 or U6 PSE sequence.
From these data, we have proposed that the U1 and U6 PSE sequences act as differential allosteric effectors of DmPBP. According to this model, when DmPBP binds to a U1 PSE DNA sequence, it adopts a conformation that allows it to recruit only RNA polymerase II basal factors during subsequent steps of pre-initiation complex assembly. On the other hand, when DmPBP binds to a U6 PSE, it adopts a conformation compatible with the recruitment of only RNA polymerase III basal factors. Biochemical and biophysical experiments are in progress to further test this model.
Selected Publications
- G. Hernandez Jr., F. Valafar, and W.E. Stumph. "Insect Small Nuclear RNA Gene Promoters Evolve Rapidly yet Retain Conserved Features Involved in Determining Promoter Activity and RNA Polymerase Specificity." Nucleic Acids Res. 2007, 35, 21-34.
- H.T. Lai, H. Chen, C. Li, K. McNamara-Schroeder, and W.E. Stumph. "The PSEA Promoter Element of the Drosophila U1 snRNA Gene is Sufficient to Bring DmSNAPc into Contact with 20 Base pairs of Downstream DNA." Nucleic Acids Res. 2005, 33, 6579-6586.
- C. Li, G.A. Harding, J. Parise, K.J. McNamara-Schroeder, and W.E. Stumph. "Architectural Arrangement of Cloned Proximal Sequence Element-Binding Protein Subunits on Drosophila U1 and U6 snRNA Gene Promoters." Mol. Cell. Biol. 2004, 24, 1897-1906.
- McNamara-Schroeder, K.J., R.F. Hennessey, G.A. Harding, R.C. Jensen, and W.E. Stumph. "The Drosophila U1 and U6 Gene Proximal Sequence Elements Act as Important Determinants of the RNA Polymerase Specificity of snRNA Gene Promoters in Vitro and In Vivo." J. Biol. Chem. 2001, 276, 31786.
- Hardin, S.B., C.J. Ortler, K.J. McNamara-Schroeder, and W.E. Stumph. "Similarities and Differences in the Conformation of Protein-DNA Complexes at the U1 and U6 snRNA Gene Promoters." Nucleic Acids Res. 2000, 28, 2771.