Question

A colleague overhears you saying that “transcription is turned on by naked DNA”, and is highly amused. Explain to your colleague the importance of naked DNA in eukaryotic transcription. Include in your answer a description of the roles of two different classes of proteins that are directly involved in exposing DNA to facilitate transcription. (15 points)

Answer 1

Naked DNA: Naked DNA refers to DNA that is not associated with proteins, lipids, or any other molecule to help protect it. Naked DNA is the result of the release of genetic information into the surrounding environment, such as from bursting cells. Gene transfer by the uptake of unprotected or “naked” DNA is known as transformation. The simplest way of a non-viral gene delivery system is by using a naked vector DNA.

The principles and mechanisms underlying transcription on naked DNA are remarkably similar between eukaryotes and prokaryotes despite the increased complexity of eukaryotic transcription machinery. The typical RNA polymerase II (Pol II) transcription cycle begins with the binding of activators upstream of the core promoter (including the TATA box and transcription start site). This event leads to the recruitment of the adaptor complexes such as SAGA (Swi/Snf) or mediator, both of which in turn facilitates binding of general transcription factors (GTFs). Pol II is positioned at the core promoter by a combination of TFIID, TFIIA, and TFIIB to form the final form of the preinitiation complex (PIC). TFIIH then melts 11–15 bp of DNA to position the single-strand template in the Pol II cleft (open complex) to initiate RNA synthesis. The carboxy-terminal domain (CTD) of Pol II is phosphorylated by the TFIIH subunit during the first 30 bp of transcription and loses its contacts with GTFs before it proceeds onto the elongation stage. Meanwhile, the phosphorylated CTD begins to recruit the factors that are important for productive elongation and mRNA processing