David A. Schneider, Ph.D.
Assistant Professor, Department of Biochemistry and Molecular Genetics
Areas of Focus: The overall goal of our lab is to characterize the molecular mechanisms that control transcription of rRNA by RNA polymerase I (Pol I) and subsequent rRNA processing events.
Ribosome abundance is directly proportional to cell growth and proliferation rates. Nucleolar size and ribosomal RNA (rRNA) synthetic activity have been used as indicators of cell transformation for more than 100 years. The overall goal of our lab is to characterize the molecular mechanisms that control transcription of rRNA by RNA polymerase I (Pol I) and subsequent rRNA processing events.
Our current interests include:
- covalent modifications that regulate the activity of "core factor," an essential transcription initiation factor for Pol I;
- mechanisms by which the conserved eukaryotic transcription elongation factor Spt4/5 influences Pol I transcription elongation;
- identification and characterization of novel transcription elongation factors that regulate rRNA synthesis rates and rRNA processing.
We use a large variety of strategies to investigate these questions including: genetic analyses (screens, in vivo rRNA synthesis assays, ChIP), biophysical analyses (mass spectrometry, in collaboration with Dr. Matthew Renfrow; UAB), biochemical analyses (in vitro enzymology of Pol I) and electron microscopy (in collaboration with Dr. Ann Beyer, University of Virginia). One example of our implementation of diverse techniques to address a fundamentally important question is shown in the accompanying figure. We used a genetic screen to identify a mutation in Pol I (A135 subunit) that rendered cells sensitive to the transcription elongation inhibitor 6-azauracil. We then purified the mutant enzyme and demonstrated its transcription elongation rate was significantly slower than wild-type. Ultimately, we used that mutant strain to demonstrate, for the first time, that transcription elongation by Pol I is functionally coupled to processing/folding of rRNA.
Our long-term goal is to fully characterize factors that regulate initiation and elongation steps in Pol I transcription. Without understanding the robust mechanisms that control and optimize transcription initiation and elongation by Pol I, the long known role for ribosome synthesis in cell transformation cannot be defined or controlled.
|Mutation of Aspartate 784 to Glycine in A135 Impairs Pol I Elongation Rate. (A) Ten-fold dilutions of yeast cells with WT A135 (NOY388) and rpa135(D784G) (NOY2172) carrying pRS316 were plated on SD -Ura medium and SD -Ura +250 μg/ml 6AU.(B) Protein sequences flanking D784 of S. cerevisiae (S.c.) A135 aligned with S.c. Rpb2, Homo sapiens (H.s.) Rpb2,
and Escherichia coli (E.c.) β. Numbering is relative to S.c. A135, and conserved acidic residues equivalent to D784 are shaded in gray.(C) Multiround in vitro transcription assays varying the concentration of UTP (left) or ATP (right) using purified WT or rpa135(D784G) Pol I.
Runoff product accumulation was normalized to the value at the highest NTP concentration (the values were as follows: for UTP, WT 6983 and rpa135 6893;
for ATP, WT 5328 and rpa135 8254 in arbitrary units) and plotted versus the varied NTP concentration. Raw data are inset in each plot.(D) Elongation rate assay measuring runoff product (763 nt) accumulation versus time after release of elongation complexes arrested at +56 (relative to transcription start site) using WT and rpa135(D784G) mutant Pol I. Raw data are shown on the left, with quantification of the amount of runoff product normalized to maximum and plotted versus time on the right.
Dr. Schneider was raised in the suburbs of Atlanta, GA. He obtained his B.S. in microbiology and genetics from the University of Georgia in 1998. As an undergraduate he studied molecular biology of bacteriophage. As a graduate student at the University of Wisconsin (1998 - 2003), he moved up the tree of life to study the effect of small molecule regulators on transcription in bacteria. For his postdoctoral studies at the University of California, Irvine (2003 - 2007), he inched further up the tree of life to study the molecular mechanisms by which RNA polymerase I is regulated in eukaryotic cells. His work continues to focus on studies in model eukaryotic cells (yeast). Dr. Schneider joined the faculty as an Assistant Professor at UAB in 2007.