Abstract
Publisher Summary This chapter presents protocols developed to study transcription antitermination and related biochemical functions of Escherichia coli (E. coli) CspE protein. The methods described can be easily adapted to study other Csp proteins as well as unrelated proteins known or suspected to interact with RNA and affect transcription termination efficiency. To test the effect of CspE on transcription antitermination in vitro, pure proteins free of RNase contamination must be obtained at high concentration. The procedures for purification of CspE are discussed. The chapter discussed both in vivo and in vitro assays. These methods are useful for testing proteins that exhibit functional or structural similarity to CspA. In E. coli, the factor-independent transcription terminator signal is encoded in the nascent RNA and consists of two essential elements, a stem loop structure followed by a stretch of U residues. CspA homologs appear to target the stem-loop structure and thus decrease termination of transcription. Impairment of either the RNA binding or RNA melting activity can affect transcription antitermination function of CspA homologs. Biochemical assays have been designed that allow one to quantitatively determine these activities.
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