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Abstract
non-coding RNAs (ncRNAs) are important players in regulating gene expression. Regulatory ncRNAs are present in all domains of life and are even found in viruses, acting as global regulators that affect gene expression both directly and indirectly at the transcriptional and post-transcriptional level. ncRNAs in bacteria are dominated by a group called small RNAs that regulate through interactions with a protein chaperone, Hfq, and base-pairing with target mRNAs. Other types of ncRNAs in bacteria include riboswitches, cis-acting ncRNAs that can respond to ligand binding, and the defense system against mobile genetic elements, CRISPR/Cas. Identification of regulatory RNAs are increasing, but many of their functions have yet to be elucidated. Structural studies of regulatory ncRNAs, through X-ray crystallography or NMR, can be used to learn about ncRNA function. Large amounts of homogenous RNA are necessary for both applications. In vitro transcription utilizing a phage RNA polymerase (RNAP), such as T7 phage RNAP, is the most common method for overproduction of RNA. Characteristics that make T7 RNAP an attractive option include that it is easily overproduced and purified, has high specificity for its promoter, and transcribes at a faster rate than bacterial RNAP holoenzyme. Although there are benefits to a T7 phage RNAP system, there are also negatives. T7 RNAP is known to transcribe RNAs with non-homogenous 5 and 3 ends. Methods developed to remedy this add time and complexity to the overall process. We propose a RNA production system that utilizes bacterial RNAP and alternative sigma factors to avoid the issues associated with T7 RNAP. The process combines an easy cloning strategy and the flexibility of producing RNA both in vitro and in vivo