All organisms have a multiplicity of integrated regulatory circuits to ensure the efficient production of ribosomes. RNA polymerase I (RNAP 1), which is responsible for the bulk of rRNA synthesis, appears to be the key element in adjusting the cellular synthetic capacity to meet, but not exceed, the demand for protein. In rapidly growing cells, RNAP I activity comprises the bulk of total transcription, where as in quiescent cells it is virtually shut-off. The long tenn objective of this work is to understand the molecular mechanisms of RNAP I transcription, and how such extremes of transcriptional activity are achieved. One of the best systems to study such a complex process is Saccharomyces cerevisiae, as it is amenable to both biochemical and genetic analysis. But the potential of the yeast system has not been realized, in large part because only recently has RNAP I transcription been reproduced in vitro. An immediate objective of this work is to characterize the trans-acting factors required for the formation of preinitiation transcription complexes using a recently developed yeast in vitro system. To facilitate the purification of these factors, single-round and rapid multiple-round transcription assays will be developed. Factors will be further purified from previously characterized chromatographic fractions using low, and medium pressure (FPLC-type columns) chromatography. The fractions, and how they interact in the initiating transcription complex, will be characterized in the following ways: 1) The fractions (and as they become more pure, factors) required for transcription complex formation will be determined by reconstituting specific RNAP I transcription with combinations of fractions. 2) The composition of the stable transcription complex will be studied directly using DNA binding assays of the fractions (alone and combinations) and indirectly by assaying activity of complexes challenged with competing/destabilizing agents such as a second RNAP I template or heparin. 3) The factor(s) regulated by the cellular growth rate will be identified, and purified, by assaying for ability to restore specific transcription to inactive extracts prepared from stationary phase cells. 4) It will be determined if other agents/treatments, known to specifically affect rRNA synthesis, function through the same mechanism as the growth rate regulation.
| He, B; Riggs, D L; Hanna, M M (1995) Preparation of probe-modified RNA with 5-mercapto-UTP for analysis of protein-RNA interactions. Nucleic Acids Res 23:1231-8 |
