Recent advances have established attenuation as a major gene regulatory mechanism employed in bacteria, their viruses, and possibly by animal viruses. We propose to continue studies of the basic features of attenuation in the trp operons of E. coli, Serratia marcescens and other bacterial species. We will study transcription pausing in the leader region of the trp operon of E. coli and determine whether pausing is responsible for the synchronization of translation and transcription that is essential to regulation by attenuation. We will examine mutationally altered transcripts to learn the structural requirements of pausing. We will characterize the paused complex and identify the subunits of RNA polymerase that interact with and recognize the RNA pause signal. We will also study the sequence and structural requirements for efficient translation initiation at the leader ribosome binding site. We will produce oligomer-directed mutations in the trp leader region to assess the role of each segment of the leader transcript in regulation by attenuation. We will analyze the base-paired RNA secondary structures that form in the trp leader transcript of Serratia marcescens to determine if these structures are consistent with secondary structure predictions and expectations based on functional studies with deletion mutants. We will also use synthetic oligomers as in vitro competitors of RNA secondary structures as a means of verifying hypothesized functions of specific structures. We will isolate additional leader mutants altered in RNA segments 1, 2 and 3 in an effort to understand how the steady-state level of read-through transcription is set for cultures growing with excess tryptophan. We will test our hypothesis that ribosome dissociation at the leader peptide stop codon, and equilibration of RNA structures 1:2 and 2:3, are the principal events responsible for setting the steady-state read-through level. Overall these studies should reveal basic features of transcription and translation and how they are employed in regulation of gene expresssion. In addition, these studies should provide insight into the significance of RNA secondary structures as signal in fundamental biological processes. We will continue investigations on translational coupling. We will determine the requirements for activation of the trpA ribosome binding site, and examine the role played in this process by the robosome that translates trpBmRNA. We will fuse trpB and trpA of E. coli and examine the effect of fusion on B and A enzymatic functions.
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