One of the fundamental questions in biology is how cells regulate their growth. Aberrations of growth regulation result in birth defects, hypertrophy and in cancer. Key to growth regulation is managing the transcription of genes tightly linked to cellular proliferation rates. Ribosomal components (ribosomal RNA, 5S RNA, and ribosomal proteins) have genes whose expression is most tightly regulated with growth. Ribosomal RNA transcription regulation, in particular, is under study as a target for arresting the uncontrolled cellular proliferation found in cancer cells. Ribosomes are complex organelles which are the site of protein synthesis. In eukaryotes, they consist of 4 RNAs (18S, 5.8S, 28S, and 5S RNA) plus about 80 proteins. Their biosynthesis involves exquisitely coordinated expression of all 80+ components, and is tightly regulated with growth in all species, from bacteria to humans. This is because in growing cells these are by far the most transcriptionally active genes in the cell; up to 85% of all transcription is from these genes. Thus from an energetic perspective alone it is crucial to properly regulate ribosome elaboration. Further underscoring their importance, there are no known human diseases of ribosome expression; these simply result in death of the embryo. However, cancer cells exhibit a very robust increase in rRNA transcription that is necessary for their uncontrolled growth. Thus, disruption of this "up" regulation is a possible target for arresting tumor growth. The ribosomal locus has also been implicated in the aging process; rRNA gene stability and nucleolar organization abnormalities directly correlate with aging in both yeast and in human genetic diseases with rapid aging phenotypes, Werner Syndrome and Bloom Syndrome.

My laboratory is studying regulation of ribosome elaboration in eukaryotic cells. The problem faced by the cell is two fold: first, keeping the correct number of ribosomes in the cytoplasm, and second, coordinating the balanced synthesis of all the 80+ components. We are studying both processes, focusing on transcription of the stable RNAs of the ribosome (5S RNA and rRNA). This is particularly interesting because 5S RNA is transcribed by RNA polymerase III, while the rRNA precursor is made by RNA polymerase I. (The mRNAs for the ribosomal proteins are transcribed by the third eukaryotic RNA polymerase, pol II.)

Our work has defined much of what we know about the mechanism of rRNA precursor transcription. Initiation requires a transcription factor (TIF-IB or SL1) which binds to the promoter's narrow groove (1) upstream and overlapping the transcription start site (2), but does not use the DNA-binding domain of its TBP subunit to do so (3,4). This complex exhibits very tight binding, the apparent Kd is 50 pM. Recent results show another factor (TIF-IE) is also required to form this complex, converting it to the "committed complex" (12). The committed complex is recognized and bound by RNA polymerase I, probably in concert with another factor, TIF-IA, to form the initiation complex. Polymerase I does not recognize DNA sequence during this binding reaction, but instead relies solely upon protein-protein interactions with TIF-IB (5, 4). DNA melting occurs next (6), but ATP beta-gamma hydrolysis is not required (7). In this area, our current focus is defining the protein-protein interactions between polymerase I and TIF-IB involved in recruitment (see below).

When starved, Acanthamoeba stops cellular growth, and rRNA and 5S RNA transcription are shut off. We were the first to show rRNA transcription is regulated by modification of RNA polymerase I or something associated with it; polymerase from transcriptionally inactive cells is unable to interact with TIF-IB to form the initiation complex (8). Recent results in yeast have shown that a specific transcription factor, Rrn3p, or the polymerase itself is modified to mediate regulation. We are currently working on the chemical nature of the polymerase or Rrn3p modification(s) and on the enzyme which modifies it, using yeast as the model system. Reviewed in (9).

Because Acanthamoeba does not have an upstream promoter element or factor, and because yeast offers such strong possibilities of genetic approaches, we have recently switched some of our work to the yeast system, Saccharomyces cerevisiae. Using this system, we are mapping the positions of each factor and polymerase subunit along the promoter, using yeast two-hybrid and classical molecular genetic approaches to investigate the role each component in rRNA transcription, and investigating the mechanism of the polymerase I associated initiation factor Rrn3p. The latter is required for initiation, and it appears its association with polymerase I is regulated, either by modification of the factor or of a key polymerase subunit. We are using biochemical and molecular genetic approaches to identify both the precise mechanism of Rrn3p and its role in initiation and regulation. The mechanism of down regulation of 5S RNA transcription by polymerase III is a separate active project (10). We are interested in how transcription by these two polymerases is coordinated. Our studies have identified changes in the level/activity/or subcellular localization of the 5S RNA-specific transcription factor, TFIIIA (11). TFIIIA is not sequestered in the cytoplasm as in amphibian oocytes, so either its synthesis or DNA-binding activity must be altered.

Additional Training and Collaborations I have been fortunate to study in the top laboratories in the world. Postdoctoral work was done with Bill Rutter at UCSF, where the multiple eukaryotic RNA polymerases were discovered at about the time of my arrival. After getting my own laboratory started, I did a sabbatical with Pierre Chambon in Strasbourg, France, where I developed one of the first in vitro transcription systems for ribosomal RNA genes. Several years later, I spent a year with Andre Sentenac in Saclay, France, honing skills necessary for the study of eukaryotic transcription. I continue collaborations with the latter group, and with Masayasu Nomura's group in Irvine, California. These, along with strong interactions with the Laybourn and Stargell labs at CSU, are especially fruitful now that we are using yeast genetics in our research.

I served for eight years on the prestigious NIH Molecular Cytology and Molecular Biology Study Sections, and continue to serve on an ad hoc basis on NIH Study Sections and Site Visits, where I have learned a great deal about grant writing and reviewing (which I teach at CSU).

1. "The fundamental ribosomal RNA transcription initiation factor, TIF-IB (SL1, factor D), binds to the rRNA core promoter primarily by minor groove contacts" Geiss, G.K., Radebaugh, C.A. and Paule, M.R., J. Biol. Chem. 272, 29243-29254 (1997).

2. "Identification of previously unrecognized common elements in eukaryotic promoters: a ribosomal RNA gene initiator element for RNA polymerase I" Radebaugh, C.A., Gong,X., Bartholomew, B. and Paule, M.R., J. Biol. Chem. 272, 3141-3144 (1997).

3. "TATA-box binding protein, TBP, is a constituent of the polymerase-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II and III transcription factors," Radebaugh, C.A., Matthews, J.L., Geiss, G.K., Liu, F., Wong, J-M., Bateman, E., Camier, S., Sentenac, A. , and Paule, M.R., Mol. Cell.Biol. 14, 597-605 (1994)

4. "Site-directed photo-cross-linking of rRNA transcription initiation complexes" Gong,X., Radebaugh,C.A., Geiss,G.K., Simon,M.N., and Paule,M.R. Mol.Cell.Biol. 15, 4956-4963 (1995)

5. "Promoter binding of eukaryotic RNA polymerase I is directed by transcription initiation factor and is DNA sequence-independent," Kownin, P., Bateman, E. and Paule, M.R., Cell 50, 693-699 (1987).

6. "DNA melting and promoter clearance by eukaryotic RNA polymerase I" Brenda F. Kahl, Han Li and Marvin R. Paule, J. Mol. Biol. 299, 75-89 (2000)

7. "Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide beta-gamma hydrolysis nor protein phosphorylation," Lofquist, A.K., Li H., Imboden, M.A. and Paule, M.R., Nuc. Acids Res. 21, 3233-3238 (1993)

8. "Transcription by RNA Polymerases I and III" Paule, M.R. and White, R. Nucleic Acids Research 28, 1283-1298 (2000)

9. "Specific transcription of an Acanthamoeba castellanii 5S RNA gene in homologous nuclear extracts," M.G. Zwick, M.A. Imboden, and M.R. Paule, Nuc. Acids Res. 19, 1681-1686 (1991).

10. "Coordinate regulation of ribosomal component synthesis in Acanthamoeba: 5S RNA transcription is down regulated during encystment by alteration of TFIIIA activity" Matthews,J.L., Zwick,M.G. & Paule,M.R. Mol. Cell. Biol.15, 3327-3335 (1995)

11. "A Novel Transcription Factor, TIF-IE, is Required for Homogeneous Acanthamoeba castellanii Transcription Initiation Factor, TIF-IB (SL1) to Form a Committed Complex" Catherine A. Radebaugh, William M. Kubaska, Laura H. Hoffman, Kristine Stiffler, and Marvin R. Paule. J. Biol. Chem. 273, 27708-27715 (1998)