The rate of ribosomal RNA synthesis varies dramatically with growth and in response to changes in the cell's environment. During rapid cell growth, e.g. neoplasia or wound healing, rRNA synthesis accounts for more than 70% of the total RNA synthesized. The genes for ribosomal RNA (rDNA) are transcribed by RNA polymerase I and are organized in clusters of tandemly repeating structures consisting of alternating nontranscribed spacers regions (NTS) and transcribed sequences (45S pre rRNA). It is central to our understanding of normal growth and tumorigenesis that the mechanisms by which these genes are regulated be understood. Studies of the promoters of vertebrate rDNA indicate that they have nearly identical functional elements with significantly different sequences. The NTS of yeast and Xenopus rDNA contain regions that can regulate rDNA expression either as major promoter elements or as enhancers. We have found that a trans-acting factor, probably not RNA polymerase I, forms a stable complex with sequences found in the NTS of rat rDNA, and that this complex then effects the rate of gene expression 10-17 fold in vitro. Experiments indicate that this effect is due to one specific region of the NTS, and this same region forms a DNA-protein complex detectable by a gel electrophoretic assay. We propose to define the specific regions of the NTS required for the enhanced rate of gene expression and to isolate the protein(s) responsible. A series of deletion and rearrangement mutants will be constructed in order to determine the orientation and distance dependence as well as the specific nucleotides that constitute the transcriptional element. Comparison of the effects of specific mutations on DNA-binding and on transcription will determine if the protein-DNA complex detected is related to transcription. The effects of protein binding on the structure of the NTS DNA will be examined in that it might be relevant to the mechanism of action of the NTS enhancer element. In vitro transcription assays will be carried out to determine if the effect on transcription is mediate through either an effect on elongation or on initiation. Both DNA-binding assays and in vitro transcription experiments will be used to determine if the factor(s) that binds to the NTS is related to, interacts with, or is the same as that which binds to the promoter. These experiments will increase our understanding of the regulation of ribosomal gene expression and the mechanism of action of enhancers in general.