Phosphorylation of transcription factors

Why is transcription important in signalling?

Transcription matters for short-term effects but, more importantly, for long-term effects that determine cell fate.

What is the general transcription machinery?

• DNA: coding strand (5′→3′) and template strand (3’→5’). The promoter sits at the start of the gene.

• RNA polymerase II is the holoenzyme, bound to general TFs; together they assemble a complete mRNA from ribonucleotides using the DNA template.

• Promoters contain TATA sequences, recognised by TBP (TATA-box binding protein).

• ~5000 genes are being transcribed in a given cell. Sequence-specific TFs bind regulatory regions; many bind together, forming a combinatorial code that tells the cell which genes to switch on.

• Mediators and TAFs bridge sequence-specific TFs to the general TF machinery.

How is the pre-initiation complex formed and how does it transition to elongation?

• TFIID binds first: TBP recognises the AT-rich TATA promoter and binds the minor groove; TAFs sit on top.

• TFIIA stabilises TFIID binding. TFIIB acts as a ruler, setting the distance from the TATA element to the start site.

• Pol II, associated with TFIIF, is recruited to the promoter-bound D-A-B complex. TFIIE and TFIIH subsequently join to complete the pre-initiation complex (PIC) → positions Pol II correctly and enables transcription initiation.

• TFIIH is a kinase: it phosphorylates Ser5 of the CTD (the heptad repeats of Pol II - YSPTSPS). This signals initiation; Pol II makes small RNAs of <100 nt = poised.

• CDK9 phosphorylates the CTD at Ser2 = signal for elongation, so full-length mRNA is made.

What is the structure of chromatin?

• Chromatin compacts the eukaryotic genome into a tiny nucleus. It is built from repeating nucleosomes: a flat disc of ~150 bp DNA looped around an octamer of histones.

• The nucleosome is a barrier to transcription, so it must be regulated and organised:

–    Euchromatin: loose; associated with active transcription (Pol II and TFs can reach the DNA).

–    Heterochromatin: tightly packed; limits access to DNA, so acts as a barrier.

• Chromatin organisation is therefore a key mechanism deciding which genes are on or off.

What is the role of cohesin?

Cohesin forms a ring essential for chromatin loops. Loops bring enhancers and promoters together; there, regulatory sequences (bound by TFs, accessed by the general machinery) control whether a gene is expressed or repressed.

What is the role of a kinase?

A kinase transfers the terminal γ-phosphate of ATP to a substrate. This causes a conformational change and creates a new interaction surface (e.g. phosphorylated receptors create docking sites for adaptors) or can activate an enzyme.

What is CREB and what is its structure?

CREB (cAMP Responsive Element Binding protein) is a TF downstream of cAMP → PKA. It is 341 amino acids long.

• Q1 – glutamine-rich

• KID (kinase-inducible domain) – contains Ser133

• Q2 – glutamine-rich

• bZIP – basic leucine zipper; mediates dimerisation and DNA binding

How is CREB activated?

• PKA phosphorylates Ser133 (KID) → conformational change → CBP (CREB binding protein) binds → complex binds the enhancer of the gene.

• Ser133 is conserved through evolution. In humans it is also conserved in CREM and ATF-1; all are members of the bZIP TF superfamily.

What is CBP?

CBP is 2441 aa. Its KIX domain recognises phospho-Ser133; its HAT domain (histone acetyltransferase) acetylates histones, opening the DNA so CREB and the transcriptional machinery can work.

How does acetylation affect DNA?

• Histones have protruding N-terminal tails, heavily modified by phosphorylation, acetylation and methylation.

• Acetylation neutralises lysine's positive charge, which weakens the histone–DNA attraction, making DNA more accessible so transcription can occur.

• HDAC removes acetyl groups, so acts opposingly to HAT.

How is CREB implicated in neurons?

CREB-containing neurons integrate different inputs and run transcription programmes that stimulate the establishment of new synapses.

• Short-term memory: no new synapses and no new proteins, so signalling does not reach the nucleus.

• Long-term memory: needs new synapses and new proteins, so signalling changes transcription of genes needed to build synapses.

What early genes are activated by CREB in long-term memory?

• Ubiquitin hydrolase: stimulates degradation of specific proteins that inhibit long-term memory.

• C/EBPs (CAAT-box Enhancer Binding Proteins): family of bZIP TFs.

• Activating Factors (AFs): interact with C/EBP, CREB1 and CREB2.

• C/EBPs and AFs (TFs and co-factors) control a second wave of transcription essential for growing new synapses.