As part of his interest in the mechanisms of regulation of the leucine tRNA multigene family in E. coli, Dr. Holmes has been involved in structure-function and regulatory analyses of the promoter of leuV, which encodes tRNALeu1, a major tRNA in the cell. A more immediate general aim is to uncover mechanisms whereby a tRNA promoter is regulated by changes in growth rate (GDR, growth-rate dependent regulation) and by the stringent response (SC, stringent control). Although much information has been accumulated concerning gene organization, structure, and function of the leucine tRNA multigene family, no clear models for regulation have emerged. It is clear from results in the last few years, mainly from Dr. Holmes' laboratory and that of Dr. Kurland, that GDR does not mean simply that synthesis of stable RNAs in general increases with increasing growth rate, but rather that some tRNAs are regulated differently from ribosomal RNAs (rRNAs) and differently from other tRNAs. Furthermore, while much is known about the metabolism of guanosine tetraphosphate (ppGpp), the major agent of the stringent response, little is known about the actual mechanism(s) by which it modulates the activities of various genes and how differently it may work with different genes. Dr. Holmes concludes that the mechanisms of action of ppGpp will eventually be found to be varied and complex. Further complication comes from conflicting findings (and interpretations) concerning the possible role of ppGpp in GDR. Dr. Holmes' studies on the in vivo regulation of tRNA operons that encode tRNALeu species, along with some preliminary in vitro studies, have yielded several very interesting findings that form the basis for the present proposal. First, he has analyzed promoter mutations that selectively abolish GDR or SC, indicating that the promoter targets for the two controls are different and suggesting that not all stable RNA genes utilize identical mechanisms for them. Second, he has identified a protein fraction that binds to a small promoter sequence that is crucial for promoter efficiency and that is required for the stringent response.That protein fraction does not bind to a mutant form of that sequence, one that also is refractory to stringent control. Third, he has generated evidence for post- initiation control by ppGpp. Finally, he has generated chromosomal gene mutations that appear to be altered in GDR, opening up the possibility of identifying the various contributors to the GDR control mechanism.
In Specific Aim 1, Dr. Holmes will analyze further various promoter sequences that he has found are important for the function of the leuV promoter. He will purify the 40 kilodalton (kd) protein that seems to be involved in the leuV stringent response, and identify promoter sequences required for optimal binding of the protein. He will try to identify other factors involved in in vitro regulation and their promoter-interactive sites, and he will be especially interested in the interaction of RNA polymerase, ppGpp, and other factors with specific promoter sequence. In the other two specific aims, Dr. Holmes will examine how post-initiation control is involved in leuV regulation and will isolate and characterize chromosomal mutations that affect leuV regulation.
