Transcript Document
Regulation of Gene Activity in Eucaryotes 1. 2. 3. 4. Transcription RNA processing mRNA transport mRNA degradation and storage 5. Translation 6. Posttranslational modulation of protein activity } Chapter 12. Transcriptional Activators in Eucaryotes Chapter 11. General Transcription Factors in Eucaryotes. Eucaryotic RNA polymerases rely upon TF for binding to promoters. For increased level and fine control… activators..specific TF bind to upstream region of promoters…enhancers Eucaryotic cells have gene-specific transcription factors (activators). 1. To increase transcription, 2. Permits cells to control expression of their genes. (stimulate or inhibit) Activators have 2 functional domains • • Transcriptional-activation domain • - DNA-binding Domains 1. Zinc-containing modules. A variety. Use 1> zinc ions to arrange protein motif so that alpha helix resides within DNA major groove. A. B. -) C. Modules containing 2 zinc and 6 cysteines such as yeast activator GAL4 2. Homeodomains. 60 aa. 3. Beta barrels. 4. bZIP and bHLH motifs. Highly basic domain and leucine zipper/HLH Transcription-activating Domains 1. Acidic domains (acid blob)- GAL4…49 aa domian with 11 acidic residues 2. Glutamine-rich domains- Sp1- 2 domains of 25% glutamine. 3. Proline-rich domains- CTF 19/84 aa proline Structure and importance unclear Protein/DNA interactions Specific interactions between bases and aa Direct Nondirect (salt bridges, through water molecules) DNA-binding domains 1. Zinc fingers. - beta/alpha 3D Structure: Finger contour- dashed line Left side of finger is alpha helix, - Zinc finger interaction with DNA Major Groove. Curve of protein matches curve of DNA helix. –wrap around. Binding between each finger and DNA binding site relies on direct aa-base interactions A. aa preceding alpha helix B. residues 2 and 3 within helix. Utility of alpha helix contacting major groove. B- bind DNA backbone and help position alpha helix Most of the DNA contacts on one strand. Most contact bases. Details: mouse Zif266immediate early protein. - 2. GAL4 protein- yeast activator that controls genes for metabolism of galactose. Each gene has a GAL4 target site upstream of start. Target sites= -. First 65 aa- DNA binding motif 65-94- similar to zinc finger (zinc and cysteine). But different…..6 cysteines and no histidines. One zinc per 3 cysteines. Recognition: - 3. Nuclear Receptors Members of this class (- Ex. Sex hormones, glucocorticoids, Vit D, thyroid hormone, retinoic acid A. DNA binding domain with 2 zinc containing modules. 4 cysteines. B. Protein protein interaction GR in inactive form in nucleus complexed with hsp. Glucocorticoid diffuses across cell membrane and enters cytoplasm. Transcriptional activation. 1. Binding domain dimerizes, making contacts with DNA. 2. 3. Binding domain a zinc module that contains 2 zinc ions. 4. -. 5. Alpha helical interaction Homeodomains: Members of the HTH family of DNA binding domains. Each domain contains: 3 alpha helixes: 2 and 3 form HTH motif, 3 is recognition helix. One helix in major groove, other across DNA at angle Engrailed homeodomain. Recognition helix fits into DNA major groove and makes specific contacts. Beta- barrels The factor dimerizes by forming a beta barrel at the interface between the 2 subunits. Shaped like a barrel, composed of anti-parallel beta sheets. Positions alpha helices into major groove. bZIP and bHLH DNA binding (b= basic region in each domain that forms the majority of the DNA binding domain) and dimerization (leucine zipper and HLH). Model for Leucine Zipper 2 alpha helices. Leucines interact like a zipper. - bHLH proteins dimerize through a HLH domain which allows basic residues of each long helix to interact with the DNA target site. One face of helix hydrophobic residues, other face charged. Length of connecting loop 12-28 amino acids Transcriptional Activation Domains: Stimulate binding of general TF to a promoter. Build up of complex. In turn, promotes polymerase binding.