Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Protein-DNA Interactions Site-specific Blackburn & Gait, p. 400-415, 418-421 Neidle, chapter Understand the basics behind HTH motif • important AAs, how protein recognizes DNA, dimerization, important contacts • examples: CAP, cro repressor, etc. Understand the basics behind Homeodomain motif • important AAs, how protein recognizes DNA, monomer, important contacts • examples: Drosophila proteins, yeast MATa2 Understand the basics behind Zinc finger motif • important AAs, how protein recognizes DNA, 3 types, important contacts • examples: Zif268, glucocorticoid receptor, GAL4 Understand the basics behind leucine zipper motif • important AAs, how protein recognizes DNA, dimerization, important contacts • examples: GCN4, jun, fos Understand the basics behind TBP binding to DNA • important AAs, how protein recognizes DNA, saddle shaped structure, important contacts Understand the basics behind RNP motif • loops of protein bind RNA • U1A protein binds toU1 snRNA Understand the basics HIV TAR RNA binding by Tat • important AAs, how protein recognizes DNA, important contacts Protein-DNA Interactions Site-specific For cell to function proteins must distinguish 1 nucleic acid sequence from another very accurately • tRNA synthetase must charge only its cognate tRNA • transcriptional activators and repressors must turn on specific genes We understand protein-nucleic acid interactions mostly from crystal structure and NMR data Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) • no stable structure by itself, needs surrounding protein sequence • first sequence-specific DNA binding protein structures solved were from proks - E.Coli CAP (catabolite activator protein) & cro repressor from phage l • both have HTH - most common sequence-specific DNA binding motif • Other examples: 434 cro, lambda repressor, 434 repressor, trp repressor • sometimes in euks - homeodomain motif (talk about later) Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) Structure well-established, ~20 amino acids Pair of helices that stack to form a V-shape (60˚ angle) Usually first helix positions the second (recognition helix) so that it projects into MAJOR groove and recognizes specific sequence Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) Structure well-established, ~20 amino acids 6 AAs out of 20 in motif help maintain correct angle Position -9 at turn between 2 helices usually Gly Positions -4, -8, -10, -15 usually hydrophobic Position -5 usually small (Ala or Gly) Motif always occurs as part of a larger structure that differs from protein to protein Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) Functions as dimer DNA sequence has twofold symmetry Recognition helix is a misnomer - both helices contact DNA Each monomer recognizes half-site Helix #2 is above MAJOR groove but its N-term is in contact with phosphate backbone Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) Repressor forms H-bonds to 4 phosphates per monomer, clamping helix #3 (recognition helix) in MAJOR groove Repressor uses backbone amides and side chain groups Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) CAP - 90˚ bend in DNA, little change to protein Ethyl-phos No pro bind Phos sensitive To DNase I Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) CAP - two kinks ~43˚ each Bases roll and unstack base pair at TG Protein-DNA Interactions Site-specific Helix-turn-helix (transcriptional regulators) CAP - Glu181 critical to form kink Electrostatics important - lots of Lys and Arg Protein-DNA Interactions Site-specific Homeodomain - Eukaryotic motif Similar to HTH? NO can fold by itself Binds euk. asymmetric homeobox sequence as monomer ~60 AA module found in: Dros. Antennapedia, Dros. Engrailed, yeast MATa2 Protein-DNA Interactions Site-specific Homeodomain - Eukaryotic motif Bind DNA by inserting long 3rd helix (recognition helix) into MAJOR groove and N-term arm into adjacent MINOR groove Protein-DNA Interactions Site-specific Homeodomain - Eukaryotic motif IMPORTANT - Asn51 makes two H bonds to A in MAJOR groove Additional links to phosphate backbone AA 47, 50 and 54 help discriminate one homeobox from another Protein-DNA Interactions Site-specific Zinc finger - Eukaryotic motif Most common euk. gene regulatory proteins have Zn fingers Zinc coordinated to Cys or His of DNA binding domain 1st discovered was from Xenopus - TFIIIA Three types: Cys2-His2 Cys4 GAL4 dinuclear cluster All use a-helices in MAJOR groove Structural data from crystallography and NMR ~30 AA domain binds Zn and folds properly Protein-DNA Interactions Site-specific Zinc finger - Eukaryotic motif Cys2-His2 3 tandemly repeated Zinc fingers 1 Zinc finger Protein-DNA Interactions Site-specific Zinc finger - Eukaryotic motif Cys2-His2 Zn staples a-helix & b-sheet together as well as forms a phobic core Zif268 contacts to G-rich DNA strand by Arg/His Protein-DNA Interactions Site-specific Zinc finger - Eukaryotic motif Cys4 nuclear receptors Two a-helix loop motifs bind as dimer to 2-fold symmetrical sequence GRE (glucocorticoid response element) in DNA is bound by dimer of glucocorticoid receptor GRE is made of 2 half-sites 5’-AGAACA XXX TGTTCT-3’ (has to be a 3-nt spacer) Protein-DNA Interactions Site-specific Zinc finger - Eukaryotic motif Cys4 nuclear receptors GRE (glucocorticoid response element) in DNA is bound by dimer of glucocorticoid receptor Protein-DNA Interactions Site-specific Zinc finger - Eukaryotic motif GAL4 • Has 3 subunits - Zn cluster, linker, dimerization region • 2 Zn ions coordinated by 6 Cys • Monomeric in solution but dimerizes upon binding 17 bp symmetrical DNA sequence with specific CCG triplets at ends Protein-DNA Interactions Site-specific Leucine Zipper (bZIP) - Eukaryotic motif • Found in certain euk regulatory DNA-binding proteins • Ex: yeast transcriptional regulatory protein GCN4 & AP1 (oncoproteins jun and fos) • Leucine zipper does not bind DNA but dimerizes proteins so they can bind DNA • Leu zipper is an amphipathic a-helix where phobics (Leu) face one side and charged AAs the other • bZIP <100 AAs, three domains (N-term regulatory, dimerization leucine zipper, basic DNA binding) • Leucine zipper - a-helix with Leu every 7 AAs, since a-helix has 3.6 AA/turn of helix all Leucines on the same face Protein-DNA Interactions Site-specific Leucine Zipper (bZIP) - Eukaryotic motif • Leucine zipper - a-helix with Leu every 7 AAs, since a-helix has 3.6 AA/turn of helix all Leucines on the same face • two basic ends form a-helices and sit in MAJOR groove • Dimerization allows protein to bind DNA in scissors-grip fashion (Y-shape) • 2-fold symmetry Protein-DNA Interactions Site-specific Basic Helix-Loop-Helix Zipper (bHLHZ) - Eukaryotic motif Protein-DNA Interactions Site-specific TATA Box binding protein - Eukaryotic motif To initiate transcription all three RNA polymerases require TATA box binding protein (TBP) which binds to MINOR groove of DNA and recognizes TATA sequence TBP 5-stranded all antiparallel b-sheet & domains connected by 7-AA linker Saddle-shaped structure Regulation of iron metabolism (eukaryotes) • The level of free iron is highly regulated in eukaryotes • Two opposing protein activities are that of the transferrin receptor which transports iron into cells, and ferritin which stores iron • The expression of each of these proteins is oppositely regulated at the translational level by the same ironsensitive factor • The iron-response element (IRE) is an RNA sequence specifically recognized and bound by the IRE-binding protein (IRE-BP) • IRE-BP binding to iron or the IRE is mutually exclusive • IRE-BP binding to ferritin mRNA inhibits translation while IRE-BP binding to the transferrin receptor mRNA stabilizes the mRNA and promotes translation IRE-BP The iron response element ferritin mRNA transferrin receptor mRNA Protein-DNA Interactions Site-specific RNA binding proteins RNP motif/domain ~90 AA sequence Example: U1A protein binds to U1 snRNA Tat-TAR in HIV Activity of Tat • Bulge loop in nascent HIV transcript is recognized by regulatory protein • Protein is Tat, trans-activator protein • Binding site is TAR, trans-activation response element • Tat-TAR interaction is required for HIV transcription • Tat stimulates full length viral RNA transcription • Without Tat, transcripts are short • With Tat, transcripts are full length Tat Binding Site • Tat protein binds to trinucleotide bulge in TAR RNA • Arginine rich basic region of Tat binds TAR • Causes a complete rearrangement in TAR conformation TAR RNA Tat Protein Protein-DNA Interactions Site-specific RNA binding proteins HIV TAR - Tat interaction Tat - Trans-activating protein (86 AA) TAR - Trans-activating RNA Protein-DNA Interactions Site-specific RNA binding proteins HIV TAR - Tat interaction TAR Structures Without Tat With Tat • Bulge closes upon binding • Other factors also bind • Changes the processivity of RNA pol • Induced fit binding Activity of Tat-TAR • • • • Tat binding recruits CyclinT-cdk9 to TAR Also recruits TFIIH to TAR Both phosphorylate the CTD of RNA pol II Improves the elongation efficiency of pol II Protein-DNA Interactions Site-specific RNA binding proteins HIV TAR - Tat interaction