Control Of Eukaryotic Gene Expression

Process by which information in genes (DNA) is decoded into protein.] Multistep process - Transcription and Translation

Transfer of genetic information from dsDNA to ssRNA (mRNA)

• 1 strand is used as a template to build up single stranded copy of template using RNA polymerase, along with ribonucleotide triphosphates (ATP, UTP, CTP, GTP). • RNA copy created that is complementary to template strand.

• Contains the signals that tell RNA polymerase to bind and start transcribing. • Promoter dictates when a gene is switched on and how efficiently.

• RNA polymerase + sigma factor makes contacts with sequences upstream of transcribed region. • Promoter has sequence elements located ~35 & 10bp upstream from transcription start site. • RNA polymerase makes initial contact with promoter sequence to form closed complex, DNA still base-paired. • RNA polymerase prises DNA open over transcription start site, gives access to template strand. • Template strand used to make RNA copy. • After ~10 nucleotides have been formed, sigma factor is released.

cis acting DNA regulatory element through which transcription is initiated and controlled.

• Transcription start-site (+1) • ~10 base-pairs upstream is -10 sequence (hexamer). • Spacer between 16-18bp, and then -35 sequence (hexamer) around -35bp. • These are consensus sequences (strongest bacterial promoters). • All signals located in small region upstream of transcription start-site.

TATA box (-30bp) Initiator (located over start-site) DPE (+30bp) BRE (next to TATA box)

Core (basal) region and regulatory region.

• C-G rich sequences, feature of just mammalian promoters • Approx. 60-70% of protein coding genes lack obvious TATA and initiator element. • Transcription initiation occurs at lower rate and at several start sites. • Associated with regions (~100-1000bp) with a high frequency of CpG islands. • In mammals, most C residues followed by a G are methylated (to 5-methyl C) • However generally C residues in CpG islands escape methylation (hypomethylated) • Important for promoter function • Methylation of CpG islands is associated with silencing (transcription switched off)

Upstream Activator Sequences, activator binding sites – increase amount of transcription initiation.

Upstream Repressor Sequences – binding site for proteins that repress/inhibit transcription initiation.

UAS/Enhancer URS/Silencer Can be proximal or many kb away. Combination of these elements dictates at what level a gene will be switched on and which signals it will respond to.

• Sequence comparison: - Used to identify TATA box - Take sequences of regions that were known to be just upstream of transcription start site and try and identify a common sequence. - No functional information about what the sequence does. • Reporter analysis: - Reporter genes encode proteins whose levels can be easily measured, enzymatically or down a microscope (GFP, luciferase, lacZ) - Amount of reporter protein provides a measure of gene expression. - Used to identify when a gene is expressed, where it is expressed/which tissue, which signals it responds to, what factors and sequences control its expression.

• RNA pol 1 * Transcribes rRNA genes. * Nucleolus • RNA pol 2 - most important * Transcribes mRNA, snRNAs, miRNAs. * Nucleus • RNA pol 3 * Transcribes tRNA, 5S RNAs, U6 RNA, 72 RNA * Nucleus

Bacterial RNA Pol: - B subunit, B prime subunit, 2 a subunits, w subunit - Similar crab claw structure Yeast RNA Pol 2: - 12 subunits - More complicated - Some direct counterparts - Similar crab claw structure

Recognise promoter regions in bacteria

Sigma 70 - housekeeping

General transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH All required to bring RNA pol to promoter and allow transcription initiation. Multi-component factors (complexes), except B.

Form a complex on TATA box, recruit RNA pol 2 to the promoter & direct initiation at start-site.

• Initiation – TFIID interacts with TATA box. • Once TFIID is assembled, TFIIA can be assembled. • … and TFIIB. Important because this is the signal that allows the recruitment of the polymerase. • RNA polymerase joins with TFIIF. • Assembly of TFIIE and TFIIH. -Equivalent of closed complex in bacteria.

• TFIIH separates the template strand at the start-site (Open Complex)- Requires ATP hydrolysis. • As Pol II begins transcribing (promoter clearance) it is extensively phosphorylated on the C-terminal domain (CTD). • The CTD is a series of repeats located at the C-terminal end of the largest (β’ homologous subunit) of Pol II. • TFIID and TFIIA may stay behind, remaining bound. • TFIIB, TFIIE and TFIIH are released. • TFIIF moves down the template with RNA Pol II.

13 TBP + TAF Binds to the TATA box Recruits TFIIB Trilobular structure - TBP in the middle. Saddle-like structure

3 Stabilises TFIID binding Anti repression function