A key mechanism for survival of toxic challenge in prokaryotes is to employ stress-specific sigma subunits that redirect RNA polymerase to transcribe genes that enhance viability. A variant of this strategy was recently shown to be conserved in the simple eukaryote S. cerevisiae (baker's yeast), in that two subunits of RNA polymerase II (pol II), RPB4 and RPB7, are selectively incorporated into pol II during stressful growth conditions, and alter transcription in a way essential for organismal survival. We have used a series of genetic strategies in yeast to isolate previously unsuspected human homologs of RPB4 and RPB7, which we have designated hsRPB4 and hsRPB7. We have performed a preliminary series of tests in yeast and in mammalian cells, and have determined that hsRPB7 can functionally interact with the pol II holoenzyme, complementing deletion of the yeast RPB7 gene: and is expressed in an unexpectedly complex manner in human cells. Similar experiments with the hsRPB4 subunit are in progress. Our goal is to determine whether hsRPB4 and hsRPB7 function similarly to their yeast counterparts in protecting pol II and potentially redirecting pol II specificity during toxic challenge, with particular focus on cellular response to exposure to agents used to chemotherapy. To this end, we will first complete preparation of a number of reagents required for the study of hsRPB4. We will then utilize yeast strategies as an initial approach to delineate the role of hsRPB4 in altering transcription in response to toxic agents. We will also use biochemical and molecular analysis of cultured mammalian cells to characterize the expression, modification, and incorporation of hsRPB4/hsRPB7 into pol II during imposition of a variety of cellular stresses, and in control cells versus cells that have become resistant to chemotherapeutic agents. Finally, we will determine how alteration of the expression pattern of his RPB4 affects transcription of a panel of genes associated with stress response. Specifically, we will test for hsRPB4/hsRPB7 interaction with protein factors known to be involved in stress response, and determine whether modifying hsRPB4 expression alters cellular viability during stress. Our ultimate goal in these studies is to determine whether modulation of the function of these proteins may be a novel method for selectively regulating cell survival during chemotherapeutic regimens.
| Toby, G; Law, S F; Golemis, E A (1998) Vectors to target protein domains to different cellular compartments. Biotechniques 24:637-40 |
