The long term objectives of this research are to elucidate the mechanisms for the regulation of ribosomal RNA synthesis. Recently, we have purified a 37bp enhancer-binding protein (E1BF) which consists of two polypeptides, and have also partially characterized a 43bp enhancer-binding protein (E2BF). Both E1BF and E2BF bind to the core promoter as well, and E1BF enhances Pol I-directed transcription from the core promoter as much as 10-fold even under nonoptimal conditions.
The specific aims of the current proposal are (a)to determine whether the two E1BF polypeptides are structurally related. (This will be done by peptide mapping following CNBr or V8 protease cleavage), (b) to investigate whether one of the polypeptides binds to the enhancer and the other to core promoter by the electrophoretic mobility shift and DNase I protection analyses, (c) to study the function of these polypeptides (d) to determine the core or consensus element(s) in the enhancer by a combination of deletion and linker scanning mutagenesis. We will explore (1) the species-specific action, (2) effect on pre-initiation complex formation, initiation and elongation reactions (3) probable existence of two domains, one binding to DNA and the other exhibiting a """"""""transcriptionally active"""""""" region and (4) binding to other enhancer elements in the spacer. We will also attempt to distinguish """"""""scanning model"""""""" from a """"""""looping model"""""""" to explain the action of the enhancer. We will then clone the gene for E1BF, express it in bacteria, study the function of the bacterially produced protein, determine the cDNA and peptide sequences, examine relationship, if any, of the gene and its product to those of other known genes and polypeptides and study the expression of this gene under different conditions such as glucocorticoid-induced stimulation in liver, serum-dependent growth, in hepatoma vs resting liver, all of which lead to up regulation of rRNA synthesis, and cycloheximide treatment and serum deprivation which result in down regulation of rRNA synthesis. Phosphorylation and dephosphorylation of E1BF and Pol I under the above conditions will also be examined. The effect of phosphorylation on the binding E1BF to the core promoter and enhancer, and on the stability of the initiation complex will also be studied. Finally, these studies win be extended to E2BF, the 43bp motif-binding factor. It is anticipated that these studies will reveal the basic molecular mechanism (s) for the regulation of ribosomal RNA synthesis, a key cellular event, in growth, tumorigenesis and following hormone treatment.
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