protein-dna interactions site-specific blackburn & gait, p. 400-415, 418-421 neidle, chapter...

Protein-DNA InteractionsSite-specific

Blackburn & Gait, p. 400-415, 418-421Neidle, 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 MAT2

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


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


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)


Helix-turn-helix (transcriptional regulators) Structure well-established, ~20 amino acidsPair 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


Helix-turn-helix (transcriptional regulators) Structure well-established, ~20 amino acids6 AAs out of 20 in motif help maintain correct angle Position -9 at turn between 2 helices usually GlyPositions -4, -8, -10, -15 usually hydrophobicPosition -5 usually small (Ala or Gly)Motif always occurs as part of a larger structure that differs from protein to protein


Helix-turn-helix (transcriptional regulators) Functions as dimerDNA sequence has twofold symmetryRecognition helix is a misnomer - both helices contact DNAEach monomer recognizes half-siteHelix #2 is above MAJOR groove but its N-term is in contact with phosphate backbone


Helix-turn-helix (transcriptional regulators) Repressor forms H-bonds to 4 phosphates per monomer, clamping helix #3 (recognition helix) in MAJOR grooveRepressor uses backbone amides and side chain groups


Helix-turn-helix (transcriptional regulators) CAP - 90˚ bend in DNA, little change to protein

Ethyl-phosNo pro bind

Phos sensitiveTo DNase I


Helix-turn-helix (transcriptional regulators) CAP - two kinks ~43˚ eachBases roll and unstack base pair at TG


Helix-turn-helix (transcriptional regulators) CAP - Glu181 critical to form kinkElectrostatics important - lots of Lys and Arg


Homeodomain - Eukaryotic motifSimilar to HTH? NO can fold by itselfBinds euk. asymmetric homeobox sequence as monomer~60 AA module found in: Dros. Antennapedia, Dros. Engrailed, yeast MAT2


Homeodomain - Eukaryotic motifBind DNA by inserting long 3rd helix (recognition helix) into MAJOR groove and N-term arm into adjacent MINOR groove


Homeodomain - Eukaryotic motifIMPORTANT - Asn51 makes two H bonds to A in MAJOR grooveAdditional links to phosphate backboneAA 47, 50 and 54 help discriminate one homeobox from another


Zinc finger - Eukaryotic motifMost common euk. gene regulatory proteins have Zn fingersZinc coordinated to Cys or His of DNA binding domain1st discovered was from Xenopus - TFIIIAThree types:

Cys2-His2Cys4GAL4 dinuclear cluster

All use -helices in MAJOR grooveStructural data from crystallography and NMR~30 AA domain binds Zn and folds properly


Zinc finger - Eukaryotic motifCys2-His23 tandemly repeated Zinc fingers

1 Zinc finger


Zinc finger - Eukaryotic motifCys2-His2Zn staples -helix & -sheet together as well as forms a phobic core Zif268 contacts to G-rich DNA strand by Arg/His


Zinc finger - Eukaryotic motifCys4 nuclear receptorsTwo -helix loop motifs bind as dimer to 2-fold symmetrical sequenceGRE (glucocorticoid response element) in DNA is bound by dimer of glucocorticoid receptorGRE is made of 2 half-sites5’-AGAACA XXX TGTTCT-3’ (has to be a 3-nt spacer)


Zinc finger - Eukaryotic motifCys4 nuclear receptorsGRE (glucocorticoid response element) in DNA is bound by dimer of glucocorticoid receptor


Zinc finger - Eukaryotic motifGAL4 • 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


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


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


Basic Helix-Loop-Helix Zipper (bHLHZ) - Eukaryotic motif


TATA Box binding protein - Eukaryotic motifTo initiate transcription all three RNA polymerases require TATA box binding protein (TBP) which binds to MINOR groove of DNA and recognizes TATA sequenceTBP 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


RNA binding proteinsRNP motif/domain ~90 AA sequenceExample: 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


RNA binding proteinsHIV TAR - Tat interactionTat - Trans-activating protein (86 AA)TAR - Trans-activating RNA


RNA binding proteinsHIV 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


RNA binding proteinsHIV TAR - Tat interaction

protein-dna interactions site-specific blackburn & gait, p. 400-415, 418-421 neidle, chapter...

Documents

dna important aas

surrounding protein

hth motif important

important contacts examples

tat important aas

important contactsunderstand

motif help

euks homeodomain motif