Exam 2 Ch 9 Study Guide

List 3 different classes of transcriptional activation domains

1) Acidic domain 2) glutamine-rich domain 3) proline-rich domain

List 3 different kinds of mechanisms of regulation of inducible transcription factors

1) Phosphorylation: a) start with no protein, synthesize it, becomes active (but takes a lot of time) b) inactive protein, phosphorylate it (kinase), becomes active c) phosphorylated inactive protein, dephosphorylate (phosphatase), becomes active 2)Ligand binding: put protein into membrane, no access to DNA protease (cleaves) becomes active 3) Inhibitor: inhibitor bound to protein unbind becomes active OR change of partner has inactive partner attached, signal comes in, activates partner, now protein is active

List 4 different classes of DNA binding domains

1) Zinc containing modules 2) Homeodomains (like helix-turn-helix motif in proks) 3) bZIP motifs (leucine zipper) 4) BHLH motifs (highly basic, so bind to phosphate group

What is a chimeric protein within the context of DNA binding proteins?

A protein with domains from a euk and a prok

Know the stick H-bond diagrams for each base

A/T mirror images, C/G mirror images Major groove = top minor groove = bottom D = donor A = acceptor

What is meant by specificity for DNA binding proteins? How would you change the specificity of helix-turn-helix proteins? How would you change a zinc finger protein?

AA residues can be positive, to bind to negative phosphate groups AA residues bind to specific bases protruding into helical grooves, bp do not open up so AA sense differences between bases in b-ped condition. Recognizes topology, 4 possibilites TA, AT, CG, GC So, AA side chains determine specificity helix-turn-helix = changing bend degree of DNA zinc finger = the 3 AA below the cys/his ring in the middle

How would you crystallize a protein?

Container with precipitate Drop of solution with protein high salt concentration in precipitate, 100's of salts, options are endless, pH, temp. changes, etc. changes crystallization low salt concentration and 1:1 ratio precipitate in protein drop, hangs over precipitate on sealed glass plate Let it sit for months vapor diffusion = protein drop hopefully crystallizes

List 3 different types of fxnal domains that can be found on a DNA binding protein.

DNA binding Domain Transcription domain Dimerization domain

List the main features of the lambda family repressors and the trp repressor. Include a list of protein-protein interactions as well as protein-DNA interactions.

Lambda family repressors: helix-turn-helix motif as DNA binding element AA residues in helix 3 & 3', lie in major groove, bind base pairs (hydrogen) protein-protein interaction between 5 & 5' helices (dimer) helices 1-5 = 1, helices 1'-5' = 2 434 & P22 are lambda repressors both have helix-turn-helix but recognize different sequences mutating ~3-5 residues changes DNA binding site = proves specificity per repressor not recognizing each base pair, but topology of H bonds, some accepting and some donating Each helix is in major groove on either side of DNA Arm of lower monomer "embraces" DNA to stabilize, with minor groove DNA bends when bound to protein/repressor Trp repressor: also a dimer requires tryptophan to bind (shifts E helix, straightens, protein can now fit into major groove) cannot recognize sequence without it Aporepressor cannot bind DNA, turns into repressor with trp. protein-protein interactions = tryptophan & helix ONE DNA bp interaction, phosphate-backbone interactions dominant

What are the key features of: a) The Gal4 family of transcription factor b) homeodomain proteins c) leucine zippers

a) Gal4 = yeast transcriptional activator for metabolism of glucose. Zinc containing family NOT zinc fingers 6 Cysteines + 2 Zn++/monomer = bimetal thiolate cluster DNA recognition = short alpha helix protruding into major groove b) homeodomain = control development of cell c) leucine zipper = coiled coil, 7 AA apart is another leucine, hydrophobic interactions of leucines "zip" up monomers into dimers. Highly BASIC domain b/c interacts with phosphate backbone

Explain the mechanism of glucocorticoid transcriptional regulation. Include what occurs in the absence and presence of the hormone.

diffuses through cell membrane, interacts with steroids/hormones = hormone-receptor complexes --> activators that bind to enhancer elements Glucocorticoid receptor is a soluble protein in cytoplasm associated with another protein = chaperone protein (hsp90), blocks nuclear localization signal = AA stretch that targets protein to nucleus, so stays in cytoplasm steroid/hormone/ligand crosses cell membrane into cytoplasm, binds to GR, chaperone falls off (allosteric change) nuclear localization signal sends GR to nucleus, DNA binding domain finds element, binds, turns on transcription

9 In one sentence, contrast orientations of lambda and trp repressors relative to their respective operators

lambda repressor binds mostly to base pairs with AA side chains recognizing topology, while trp repressor binds mostly to phosphate backbone, with presence of tryptophan activating it.

What is a homeodomain?

like helix-turn-helix motif of proks 60-70 AA 3 helices, 1 lies in major groove, AA side chain-bp interaction control developmental fate of cell mutations = cell develops into another cell type fruit fly --> should have antenna, but have legs because of antennapedia phenotype mutation

What constitutes the zinc finger in a DNA binding protein?

proteins bind to each other on different DNA strands (trans), creating "loops"/looping tetrahedral within major groove, "C" shape 2 cysteines 2 histidines Zn++ in middle linking AA together loop between cys-->his typically 3 fingers half = cysteine = B-sheet half = histidine = alpha-helix each finger interacts with 3 bases