William E. Stumph
Research Interests: Regulation of Gene Expression, Protein-DNA
Interactions, Assembly of the Transcription Apparatus
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 (mRNA)
splicing (the removal of intron sequences from mRNA 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 mRNAs), but these Pol II-transcribed snRNA genes contain no
introns and they lack the TATA sequences commonly found upstream of mRNA
transcription units. In contrast, U6 snRNA genes contain TATA boxes 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. In the fruit fly Drosophila melanogaster, a 21-base-pair-long
cis-acting sequence, termed the proximal sequence element, or
PSEA, is uniquely required for the
transcription of both classes of snRNA genes. The PSEA specifically interacts
with a multi-subunit protein termed the small nuclear RNA activating protein
complex, DmSNAPc. DmSNAPc is required for the transcription of U1 and
U6 snRNA genes by RNA polymerases II and III respectively. Our goal is to
understand how the same protein can in one case recruit RNA polymerase II (for
U1 transcription) but in another case recruit RNA polymerase III (for U6
transcription). In order to answer this question, we apply various biochemical
and molecular biological techniques to the study of the Drosophila system.
The U1 and U6 PSEA sequences are identical at 16 of
the 21 positions. However, we have shown that the 5 nucleotide differences
between the U1 and U6 PSEAs are sufficient to determine the RNA polymerase
specificity of the U1 and U6 gene promoters. Furthermore, by site-specific
protein-DNA photocrosslinking techniques, we have determined that DmSNAPc
consists of at least three distinct polypeptide chains that contact the DNA of
the PSEA. Significantly, the data also reveal that the precise contacts made
between the protein and the DNA are different depending upon whether DmSNAPc is
bound to a U1 or U6 PSEA sequence.
From these data, we have proposed that the U1 and U6
PSEA sequences act as differential allosteric effectors of DmSNAPc. According
to this model, when DmSNAPc binds to a U1 PSEA 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 DmSNAPc binds to a U6 PSEA, it adopts a conformation compatible with the
recruitment of only RNA polymerase III basal factors. This is illustrated
schematically in the figure below.
We are currently making mutations in these various
subunits to identify domains within the proteins that are required for complex
assembly, DNA binding, and the activation of transcription. One goal is to
identify mutations that will result in the loss of activation of transcription
by one RNA polymerase but not the other. In this way we hope to identify
domains within the proteins that are specifically required for transcription by
one RNA polymerase but not the other.
- Kim MK, Kang YS, Lai HT, et al.,
Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters
Molecular and Cellular Biology 2010, 30 2411-2423.
- Hung KH, Titus M, Chiang SC, et al.,
A Map of Drosophila melanogaster Small Nuclear RNA-activating Protein Complex (DmSNAPc) Domains Involved in Subunit Assembly and DNA Binding
Journal of Biological Chemistry 2009, 284 22568-22579.
- Lai HT, Kang YS, Stumph WE,
Subunit stoichiometry of the Drosophila melanogaster small nuclear RNA activating protein complex (SNAPc)
FEBS Letters 2008, 582 3734-3738.
- Barakat NH, Stumph WE,
TBP recruitment to the U1 snRNA gene promoter is disrupted by substituting a U6 proximal sequence element A (PSEA) for the U1PSEA
FEBS Letters 2008, 582 2413-2416.
- Hernandez G, Valafar F, Stumph WE,
Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity
Nucleic Acids Research 2007, 35 21-34.