Final answer:
T7 RNA polymerase solves the '5' problem' by using a hairpin-shaped promoter on the DNA template which guides the initiation of RNA synthesis. To terminate transcription, the DNA template is linearized at a specific point using a restriction enzyme, causing the robust T7 polymerase to dissociate from the DNA.
Step-by-step explanation:
The question addresses how T7 RNA polymerase overcomes the challenge of initiating RNA transcription at the 5' end of DNA, referred to as the '5' problem.' This enzyme originates from the T7 bacteriophage and is commonly utilized in research labs for in vitro transcription.
T7 RNA polymerase works by recognizing a specially structured, 19-nucleotide long promoter on the DNA, which folds into a hairpin shape, and this structure is crucial for the polymerase to successfully initiate transcription. This promoter sequence must be present upstream of the DNA template in a cloning vector. Because T7 RNA polymerase is highly robust, to end transcription at a specific site, the DNA template needs to be linearized by cutting with a restriction enzyme, causing the polymerase to fall off the DNA template at the cut site, which effectively terminates the transcription process.
For instance, the ykkCD sensor RNA in a cloning vector utilizes the BamHI restriction endonuclease to linearize the DNA at the desired point for transcription termination. The RNA that is synthesized by T7 RNA polymerase mirrors the top strand of the DNA, where thymine (T) is replaced by uracil (U) in RNA.
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