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 intervening 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
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
We have cloned the genes that code for the three
subunits of DmSNAPc (DmSNAP190, DmSNAP50, and DmSNAP43).
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 epitopes that
are specific for each polymerase.
Zamrod, Z., C.M. Tyree, Y. Song, and W.E. Stumph.
In vitro transcription of a Drosophila U1 snRNA gene
requires TATA box-binding protein and two proximal
Cis-acting elements with stringent spacing requirements.
Mol. Cell. Biol. 13:5918-5927 (1993).
Wang, Y., and W.E. Stumph. RNA polymerase II/III
transcription specificity determined by TATA box orientation.
Proc. Natl. Acad. Sci. USA 92:8606-8610 (1995).
Wang, Y., R.C. Jensen, and W.E. Stumph. "Role
of TATA Box Sequence and Orientation in Determining
RNA Polymerase II/III Transcription Specificity."
Nucleic Acids Res. 24, 3100-3106 (1996).
Su, Y., Y. Song, Y. Wang, L. Zhan, L. Jessop, and
W.E. Stumph. "Characterization of a Drosophila
PSE-binding Factor Involved in Transcription of Small
Nuclear RNA Genes." Europ. J. Biochem. 248, 231-237
Jensen, R.C., Y. Wang, S.B. Hardin, and W.E. Stumph.
"The Proximal Sequence Element (PSE) Plays a
Major Role in Establishing the RNA Polymerase Specificity
of Drosophila U-snRNA Genes." Nucleic Acids Res.
26, 616-622 (1998).
Wang, Y. and W.E. Stumph. "Identification and
Topological Arrangement of Drosophila Proximal Sequence
Element (PSE)-binding Protein Subunits that Contact
the PSEs of U1 and U6 snRNA Genes." Mol. Cell.
Biol. 18, 1570-1579 (1998).
Hardin, S.B., C.J. Ortler, K.J. McNamara-Schroeder,
and W.E. Stumph. "Similaritites and Differences
in the Conformation of Protein-DNA Complexes at the
U1 and U6 snRNA Gene Promoters." Nucleic Acids
Res. 28, 2771-2778 (2000).
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
Small Nuclear RNA Gene Promoters in Vitro and In Vivo."
J. Biol. Chem. 276, 31786-31792 (2001).
Li, C., G.A. Harding, J. Parise, K.J. McNamara-Schroeder,
and W.E. Stumph. “Architectural Arrangement
of Cloned Proximal Seqeunce Element-Binding Protein
Subunits on Drosophila U1 and U6 snRNA Gene Promoters.”
Mol. Cell. Biol. 24, 1897-1906 (2004).