Question

Q8. [10 marks]

Many molecules are involved in providing stability to the Pre-Initiation Complex (PIC) in eukaryotes. Describe:

1. The steps involved in stimulating initiation of transcription from a silent gene
2. How the molecules interact to provide stability to the PIC
3. How the polymerase escapes from this PIC to allow the transcript to elongate

Answer 1

a. The pre-initiation complex (PIC) is a large complex of proteins necessary for gene transcription in eukaryotes and archaea. The PIC aids in the correct positioning of RNA polymerase II on the transcription start site, produces DNA denaturation, and places DNA at the active site of RNA polymerase II for transcription to occur.

This complex is normally composed of six general transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.

In 2007, Roger D. Kornberg proposed the following pre-initiation complex model in promoters with TATA boxes:

1. TATA-binding protein (TBP, a subunit of TFIID) binds to the promoter, generating a strong DNA fold. This folding causes DNA to envelop RNA polymerase II and the C-terminal domain of TFIIB. TBP binds to the TATA box so that the space between the TATA box and the transcription start site (typically about 25-30 base pairs) ensures that RNA polymerase II is positioned properly on the promoter.
2. The N-terminal domain of TFIIB positions DNA adequately for entry into the active site of RNA polymerase II.
3. TFIIE binds to the complex and recruits TFIIH.
4. TFIIH subunits that have ATP-ase and helicase activity generate negative helical tension in DNA.
5. This tension causes the denaturation of the DNA and allows the formation of the transcription bubble. TFIIF binds to the coding strand, denatures the DNA, and keeps the denaturation bubble open.
6. The non-coding strand of DNA can then fold and enter the active site of RNA polymerase II.
7. If transcription progresses more than 6 bases, TFIIB is displaced and RNA polymerase II escapes from the promoter region to already transcribe the rest of the gene.

b. There are different ways in which the molecules interact to provide stability to the PIC:

1. In the enzyme binding domain are specifically targeted groups of hydrogen bridge donors and acceptors that interact with similar groups.
2. Ionic interactions between the negative charges of the DNA molecule and the basic residues of the binding protein domain
3. Hydrophobic interactions between the methyl group of thymine and apolar groups on the surface of the enzyme.

c. The binding of the polymerase in a suitable place causes a local unwinding of the DNA (favored by the high content of AT pairs) about the bases of -10 to +5. This structure called an "open promoter" is very stable. Once the open promoter is formed, the enzyme begins to slide on the DNA, until it reaches the first base to start the transcription.