A concerted series of in vivo and in vitro experiments will be used to identify cis-acting DNA sequences and trans-acting proteins involved in the transcription and regulation of Escherichia coli ribosomal RNA synthesis. In vitro transcription experiments will be used to carefully confirm and extend preliminary data which suggests that the NusA protein of E. coli is sufficient to cause antitermination of ribosomal RNA transcription. In vitro transcription experiments will also be used to identify and purify E. coli proteins affecting transcription efficiency, regulation, and antitermination. Recombinant DNA methods will be used to develop new assay systems capable of detecting changes in transcription frequency, regulation, and antitermination. These new assay systems are required due to the inappropriateness of existing assay systems for measuring synthesis rates during the growth conditions important to studies of ribosomal RNA synthesis. These assay systems will be used to characterize cis-acting mutations in ribosomal RNA operons to be constructed in vitro or selected in vivo. An assay system for measuring feedback regulation of ribosomal RNA synthesis will also be developed and used to characterize mutations made in vitro. Methods to select cis-acting mutations defective in feedback regulation will also be developed and employed to identify the regions involved in this regulatory response.
|Hargrove, M S; Brucker, E A; Stec, B et al. (2000) Crystal structure of a nonsymbiotic plant hemoglobin. Structure 8:1005-14|