The primary goal of this research is to elucidate the molecular mechanisms involved in two types of regulation of amino acid biosynthesis in bacteria. The first type of mechanism is gene or operon specific and regulates amino acid biosynthesis with respect to the cell's need for a particular amino acid. In the case of histidine biosynthesis, which we study as a model system, the cell senses histidine sufficiency or deficiency through the amount of charged tRNAHis. In addition, charged tRNAHis must contain two pseudouridine base-modifications in the anticodon region in order to repress the histidine operon. The regulatory mechanism through which charged/modified tRNAHis acts involves translational-control-of-transcription-termination in the regulatory region of the operon. We are particularly interested in applying functional and physiological tests of this mechanism which thus far has been based on DNA sequence analysis of the his operon control region. We are examining the role of the pseudouridine tRNA base modifications on translational control (and translational fidelity), and other effects that influence the kinetics of translation of the his operon control region. The second type of mechanism involves the unusual nucleotide guanosine 5'-di-phosphate 3'-diphosphate (ppgpp). This molecule appears to be part of a super-control system that adjusts many cellular processes in response to the cell's need for amino acids in general. We are attempting to identify and characterize the components with which ppGpp interacts to increase transcription of the histidine biosynthetic operon. We are isolating mutations of the his promoter that effect ppGpp control and will determine the specific target sequences involved. We are attempting to isolate mutations in RNA polymerase genes and genes for other protein factors potentially involved in ppGpp control. These mechanisms of specific and general amino acid regulation are being pursued through use of genetic/physiological techniques (transposable genetic elements, gene fusions), DNA sequence analysis, and cell-free systems that measure transcription and translation as indicators of gene regulation. It is anticipated that generalizations arising from these studies of prokaryotic translational controls and super-controls may apply to eukaryotic gene regulatory mechanisms.
