The tryptophan (trp) operon of E. coli consists of 5 contiguous structural genes and their associated control elements. Studies utilizing current techniques in microbial genetics, nucleic acid chemistry, and protein chemistry are proposed in 3 main areas. (1) Structure and function of transcription termination sites. The sequences of 3 trp termination sites are known: trp a (the attenuator) near the 5' end of the operon, and trp t and trp t', the tandem terminators near the 3' end. Experiments will characterize termination at these sites both in vivo and in vitro, with regard to their dependence on RNA polymerase, rho termination factor and nusA protein, the nucleotide sequence, and potential for secondary structures. The important elements of rho-independent termination will be investigated for the case of rho-dependent termination. Construction and cloning of a synthetic rho-dependent termination site will be tried. (2) Coupled interactions in termination of transcription. The role of nucleoside triphosphate hydrolysis in rho-dependent termination is unknown, but may be required to release the completed transcript from the DNA template strand. Introduction of base-analogs into the RNA or DNA, to weaken or strengthen particular pairings, should reveal information about this aspect of the mechanism. In mutational polarity, termination involves rho factor as well as the ribosome, though whether directly or indirectly is not known - experiments to probe the extent of these interactions will include characterizing """"""""pause"""""""" sites within genes, and seeking other protein factors. (3) Protein-nucleic acid interactions at regulatory sites. Experiments to probe the accessibility or protection of regions in each of the 3 trp termination sites at both the RNA and DNA level will be attempted, utilizing enzymatic and chemical approaches. Crosslinking studies will be initiated on the interactions between RNA polymerase, rho factor, template DNA, and the RNA transcript, in conjunction with protein chemical investigations of the domain structure of rho and its requirements for function, to elucidate structure/function relationships between protein and nucleic acid. The basic principles involved in these complex mechanisms in prokaryotes will probably be applicable to the control of gene expression in higher organisms. Since many diseases are the result of regulatory mechanisms gone awry, in the long run a detailed molecular understanding of these processes should contribute to the development of solutions to these health problems.
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