(a) Sketch out a basic transcription unit and illustrate the RNA polymerase II associated assembly of a transcription initiation…

(a) Basic transcription unit and assembly of the RNA polymerase II initiation complex

The labelled diagram should show a DNA segment with the coding strand drawn 5′ to 3′ above the template strand, and:

• distal enhancers (and silencers), which may lie far upstream or downstream;
• the promoter: proximal elements (CAAT box at about −75, GC box at about −90) and the core promoter (BRE and TATA box at about −25 to −30, the initiator Inr around +1, sometimes a downstream element DPE at about +30);
• the transcription start site (+1), then the 5′ UTR, exons and introns, and the 3′ UTR;
• the polyadenylation signal (AATAAA) and the termination region downstream.

Assembly of the pre-initiation complex (PIC), to be drawn in order at the promoter:

1. TFIID binds the core promoter: its TBP subunit sits in the minor groove at the TATA box and bends the DNA, while the TAFs recognise Inr and DPE (many promoters have no TATA box and depend on these).
2. TFIIA stabilises TBP on the DNA, and TFIIB binds TBP and the BRE and fixes the start site.
3. RNA polymerase II, brought by TFIIF, joins the complex.
4. TFIIE joins and recruits TFIIH.
5. TFIIH uses its ATP-dependent helicase (XPB) to melt the DNA at the start site, forming the open complex. Its kinase CDK7 phosphorylates Ser5 of the repeated heptapeptide YSPTSPS in the C-terminal domain (CTD) of the polymerase, which leaves the promoter and begins elongation; later CDK9 (P-TEFb) phosphorylates Ser2 for productive elongation.

Activators bound to enhancers reach this complex through the Mediator, while histone acetyltransferases and chromatin remodellers open the nucleosomes over the promoter.

(b) Constructing a recombinant cDNA from mRNA

The labelled diagram should show these steps in order, as a flow chart:

1. Isolation of mRNA: poly(A)⁺ mRNA is purified from cells that express the gene, by oligo(dT)-cellulose affinity chromatography.
2. First strand: an oligo(dT) primer anneals to the poly(A) tail, and reverse transcriptase (RNA-dependent DNA polymerase) copies the mRNA into DNA, giving an mRNA–cDNA hybrid.
3. Second strand: RNase H nicks the RNA, DNA polymerase I uses the RNA fragments as primers to make the second strand, and DNA ligase seals the nicks. (Alternatively, alkali removes the RNA and the hairpin at the 3′ end of the first strand primes the second strand; S1 nuclease then cuts the loop.)
4. Ends and linkers: the double-stranded cDNA is made blunt-ended with T4 DNA polymerase or the Klenow fragment, and synthetic linkers or adaptors carrying a restriction site (for example EcoRI) are ligated to it and cut to give cohesive ends.
5. Ligation into a vector: T4 DNA ligase joins the cDNA to a plasmid or λ phage vector cut with the same enzyme, giving the recombinant cDNA.
6. Cloning and screening: the vector is introduced into E. coli (by transformation, or by packaging λ DNA), recombinant clones are selected on antibiotic or blue–white medium, and the clone of the gene is picked with a labelled probe. A collection of such clones is a cDNA library.

Because cDNA has no introns, it can be expressed in bacteria, which cannot splice RNA.

(c)(i) Intrinsic mechanism of apoptotic induction

The intrinsic (mitochondrial) pathway is triggered inside the cell by DNA damage, oxidative stress, growth-factor withdrawal or ER stress.

1. The stress activates BH3-only proteins (Bim, Puma, Noxa).
2. These inhibit the anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 and activate Bax and Bak, which oligomerise in the outer mitochondrial membrane.
3. Mitochondrial outer membrane permeabilisation releases cytochrome c and Smac/DIABLO, an inhibitor of IAPs, into the cytosol.
4. Cytochrome c binds Apaf-1 with dATP or ATP; seven Apaf-1 molecules form the wheel-shaped apoptosome, which activates procaspase-9.
5. Caspase-9 activates the executioner caspases-3, -6 and -7.
6. These cleave proteins such as lamins and free the DNase CAD from ICAD, so the DNA breaks between nucleosomes; the cell fragments into apoptotic bodies, which phagocytes engulf.

(c)(ii) Principle of DNA fingerprinting

DNA fingerprinting (Alec Jeffreys, Leicester, 1984) rests on the tandem repeats of non-coding DNA: minisatellites or VNTRs (repeat unit of about 10–60 bp) and microsatellites or STRs (units of 1–6 bp). The number of repeats at each locus differs greatly between individuals, except identical twins, and is inherited, half from each parent.

Procedure: DNA is cut with a restriction enzyme (RFLP), or the repeat loci are amplified by PCR; the fragments are separated by gel electrophoresis, blotted onto a nylon membrane (Southern blot), hybridised with a labelled repeat probe and seen as a band pattern. A match with a reference sample identifies a person in forensic work, and every band of a child must come from the mother or the father in paternity tests.