Transcription Factors and Transcriptional Regulation

Categories of Transcription Factors
DNA Binding Domains in Transcriptional Regulators

Zinc Finger Domain

  • Zinc finger: ~30 amino acids, alpha helix, two beta strands around a central zinc ion.

  • Zinc interacts with 2 cysteines and 2 histidines (Cys2His2).

  • Multiple zinc fingers insert alpha helices into DNA major groove.

  • TF (transcription factor) containing Zinc finger domain - C2H2 or C4.

Homeodomain

  • Eukaryotic DNA binding domain; monomeric helix-turn-helix structure.

  • Helix 3 fits in major groove, N-terminal arm contacts minor groove.

  • TF-containing homeodomain - Containing Helix-turn-helix

Leucine Zipper (bZIP)

  • bZIP proteins: long alpha helices (60 amino acids) with leucine residues that "zip" together.

  • Helices splay at N terminal, sit in DNA major groove.

  • TF-containing leucine Zipper (bZIP)

Helix-Loop-Helix (bHLH)

  • bHLH proteins: coiled-coil, helix in major groove.

  • Four helices joined by a loop, forming a four-helix bundle.

  • TF-containing helix-loop-helix (bHLH)

Beta Sheets

  • Beta sheets and loops mediate DNA recognition.

  • p50/p65 (NF-κB) are largely beta sheet.

  • TF- beta sheet with loops MetJ regulate bacteria methionine regulon etc expression

DNA Sequence Recognition

  • Regulatory proteins recognize specific DNA sequences non-covalently.

  • Bases exposed in major and minor grooves offer different chemical groups (H bond donors/acceptors, methyl).

  • Minor groove variability is less (T-A = A-T, G-C = C-G).

  • Regulatory proteins interact primarily through the major groove.

Interactions Between Protein and DNA

  • Asparagine forms two hydrogen bonds with adenine.

  • Alpha helices and beta sheets fit in the major groove; minor groove interaction occurs when DNA is distorted.

  • Alpha helices and beta sheets fit well into the DNA major groove but tend to interact with the minor groove only when the DNA is distorted, like when TBP binds.

Promoter Recognition in Bacteria and Eukaryotes

  • TFIID binds TATA box via TBP, inducing DNA distortion and unwinding.

  • TAFs mediate recognition of INR and DPE.

  • TFIIB recognizes BRE, determines transcription direction; similar to bacterial sigma factor.

  • TFIIA stabilizes TBP-DNA; TFIIE/TFIIH bind (TFIIH unwinds DNA).

DNA-Binding Motifs

  • DNA-binding motifs tailored to fit DNA; some universal.

  • Helix-turn-helix is common.

  • Many are dimers with recognition helices spaced 3.4 nm apart.

Electrostatic Surfaces

  • Electropositive surfaces (blue), electronegative (red).

  • DNA backbone is negatively charged.

  • Positive amino acids (lysine, arginine) interact favorably with negative phosphates.

  • DNA-interacting domains have lysine, arginine, serine, and tyrosine.

Signaling Cascades and Regulation of Transcription

  • Gene expression changes with conditions; signal cascades lead to transcriptional changes.

  • Nuclear receptors respond to effectors (hormones); have DNA and ligand binding domains.

  • Ligand binding induces conformational change, recruits co-repressors/co-activators.

  • A nuclear receptor outside the nucleus may only be able to enter when bound to a ligand

NF-κB Cascade

  • NF-κB (p50/p65) is important in immune responses.

  • In unstimulated cells, I-κB holds NF-κB in the cytoplasm.

  • Infection activates I-κB kinase, phosphorylating I-κB.

  • Phosphorylated I-κB is ubiquitinated, degraded by proteasome.

  • NF-κB moves to the nucleus, activates transcription.

Transcriptional Silencing and Imprinting

  • Maternal chromosome: CTCF binds ICR, enhancer stimulates H19, blocks IGF2.

  • Paternal chromosome: CTCF cannot bind, IGF2 transcribed, H19 methylated (inhibited).

  • Failure of imprinting leads to Beckwith-Wiedemann syndrome.

  • Transcription is thought to be mediated by looping of the DNA, and determined by the access of blocking of enhancer bound proteins.

  • Chromosome 11p15.5 Imprinting control region.

Ume6 and Transcriptional Repression

  • Ume6 responds to nutritional cues in yeast.

  • Enough N/C: Ume6 binds DNA, recruits co-repressors (Sin3, Rpd3, Isw2).

  • Rpd3 (histone deacetylase) promotes compact chromatin.

  • Isw2 (nucleosome remodeling enzyme) alters chromatin.

  • Ume6 phosphorylated without N/C, Sin3/Rpd3 dissociate, Ime1 recruited.

MeCP2 and Rett Syndrome

  • Methylated DNA recruits methyl-binding domains.

  • MeCP2 binds methylated DNA, recruits Sin3A (histone deacetylase).

  • MeCP2 represses human genes; mutations cause Rett Syndrome.

  • Syndrome symptoms: autism-like, slow head growth, abnormal movement, loss of social interaction.

  • This syndrome is primarily found in girls because MeCP2 is on the X chromosome. Males have only one X chromosome and most often do not survive a pregnancy. Females would have one mutant and one good copy.

Transcriptional Silencing and RNA Interference

  • Transcriptional silencing at fission yeast mating loci is mediated by RNA interference.

  • dsRNAs are cleaved to 21bp fragments, incorporated into RITS complex.

  • RITS recruits histone deacetylase and methylase, creating heterochromatin.

  • Methylated H3 tails recruit Swi6, maintaining heterochromatin.

Transcriptional Silencing and Chromatin Structure

  • Transcriptional silencing: large chromosomal regions not transcribed.

  • Due to changes in chromatin structure: euchromatin (active), heterochromatin (silent).

Evaluation of Candidates

  • 4 factors: Oct3/4, Sox2, c-Myc, Klf4

  • iPS cells (Induced pluripotent stem cell)

Myc Protein

  • Myc protein belongs to Myc family of transcription factors, which also includes N-Myc and L-Myc genes.

  • Myc family of transcription factors contain bHLH/LZ (basic Helix-Loop-Helix Leucine Zipper) domain.

  • Myc protein, through its bHLH domain can bind to DNA, while the leucine zipper domain allows the dimerization with