Protein-DNA Interactions Site-specific Blackburn & Gait, p , 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 MAT  2 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  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 MAT  2

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  -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  -helix &  -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  -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  -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 -  -helix with Leu every 7 AAs, since  -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 -  -helix with Leu every 7 AAs, since  -helix has 3.6 AA/turn of helix all Leucines on the same face two basic ends form  -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  -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 iron- sensitive factor

The iron response element 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 ferritin mRNA transferrin receptor mRNA IRE-BP

Protein-DNA Interactions Site-specific RNA binding proteins RNP motif/domain ~90 AA sequence Example: U1A protein binds to U1 snRNA

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-TAR in HIV Activity of Tat 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 TatWith 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