Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System

Abstract

Publisher Summary This chapter describes methods to examine the role of RNA structure plays in transcription attenuation, using studies Bacillus subtilis trpEDCFBA operon as a model system. Transcription attenuation mechanisms modulate the extent of transcription read-through past termination signals, thereby regulating expression of the downstream genes. The untranslated trp leader transcript can form four RNA secondary structures that participate in the attenuation mechanism: the 5ʹ stem-loop, the pause hairpin, the antiterminator, and the terminator. RNA secondary structure can be predicted by comparative sequence analysis if reliable secondary structures already exist for several homologues, or it can be predicted by folding programs such as mfold, which rely on free energy minimization. The chapter discusses the biochemical determination of RNA secondary structure. Once formation of an RNA secondary structure is verified biochemically, it is necessary to establish that the structure participates in transcription attenuation. This can be accomplished through the use of genetic and biochemical approaches. Two different in vitro transcription assays are generally used to determine if an RNA structure participates in transcription attenuation.