Lecture 9: DNA-binding Proteins Flashcards
Sequence-specific DNA-protein recognition and DNA-binding proteins
-certain sequences bind with higher affinity than others
-recognize base-specific interactions
-altered DNA structure
-involves binding major groove
Base-specific interactions
H donor, acceptor, nonpolar groups of bases
DNA-binding proteins and nonspecific interactions
-recognize interactions of DNA phosphate backbones and sugars
-involves binding minor groove
DNA-protein complex involving induced fit-binding
-can have sequence specificity
-when binding alters DNA structure so that it is not B-form
-favoribility of induced fit depends on sequence
DNA-binding proteins
-histones
-transcription factors
-polymerases and nucleases
Nucleosome
-unit of chromatin (beads on a string)
-146 bp DNA in 1.65 turns wound around histone octamer
Histone octamer
-DNA wraps almost twice around core = 2x the natural curvature of B-form DNA
-2 copies of each H2A, H2B, H3, H4
Energy required to deform DNA
from electrostatic interactions between DNA and histones
Linker DNA of 20-90bp
occurs between histones
Histone side chains
-interact with DNA-sugar-phosphate backbone
-ARG/LYS residues insert into minor groove
-contact areas are spaced to bend the DNA around histone core
N-terminal tails of histones
-protrude from core
-become sites for acetylation to regulate gene expression
Histone deacetylases (HDAC)
-cancer targets
-inhibited by SAHA
SAHA
-first FDA approved HDAC inhibitor
-clinically effective treatment of some cancers
DNA coiling around histone core
-negative supercoil created from one wrap around histone
-compensating positive supercoil forms outside of core then is relaxed by topoisomerase
=one (net) negative supercoil
Lk in DNA wrapping
Lk is constant because ends are fixed as if circular
DNA sequence dependence for location of nucleosomes
-some exist based on energetic preferences for the bending required to form the nucleosome particle
helix-turn-helix (HTH) motif
-20 aa long binding motif
-formed by 2 helices connected by short turn
-second helix is recognition helix that binds sequence-specifically in major groove
Zinc Fingers (ZnFs)
-30 aa
-small modules with zinc playing structural role
-Zn2+ ion coordinated by 4 Cys or 2 Cys and 2 His residues
-often occur as tandem repeats with 2+ fingers
-deisgned to bind targeted DNA sequences with ultimate goal of therapeutics
ZnF amino acid domain
-30aa
-2 stranded antiparallel beta sheet and short alpha helix
-a-helix makes sequence-specific contacts along major groove
-sequence-specific but can also recognize RNA and other proteins
ZnF modules
-structurally diverse: 8 fold groups
-present in 1000 different protein
Basic region-leucine zippers (bZIP)
-leucine residues every 7th position in a-helix
-form dimers with coiled coil structure
-basic region (arg and lys residues) binds to major groove
-basic aas interact with phosphate backbone of DNA through electrostatic interactions and also the DNA bases through H bonding
bZIP examples
-fos and jun
-cAMP response element-binding protein (CREB)
Helix-loop-helix (HLH) motif
-2 amphipathic a-helices connected by a loop
-forms dimers
-dimerization domain has 4-helix bundle
-extension of one of the a-helices from DNA binding domain binds major groove of DNA
HLH transcription factors
-myoD
-myc
-max
Transcription factors
-proteins involved in process of converting/transcribing DNA into RNA
-seq-specific
-controls rate of transcription by promoting or blocking recruitment of RNA polymerase
-utilize wide range of DNA-binding structural motifs
Transcription factor-DNA interactions
-common interactions between dimeric proteins and palindromic sequences
-binding often leads to conformational changes in protein and DNA
Transcription
-use DNA to make RNA
-highly regulated
-off=DNA. condensed into nucleosomes
-on=transcription factors activating transcription
Regulation by transcription factors
-bind either promoter or enhancer regions of DNA
-recruit coactivator/corepressor proteins to the complex
-stabilize/block RNA polymerase
-catalyze de/acetylation of histones
Transcription factor function
-basal level transcription
-development
-signaling
-cell cycle control
Regulation of transcription factors
-may be de/activated by ligand binding to ‘sensing’ domain (eg hormone receptors)
-by chemical modification (eg STAT proteins must be phosphorylated to bind to DNA)
-by interaction with coregulatory proteins or with other TFs (dimerization)
Eukaryotic TFs
-modular structure (multiple domains)
-ex: Creb
CREB
-DNA binding domain (bZIP)
-activation domain (Q1 and Q2)
-Protein interaction domain (KID)
DNA binding domain (bZIP)
-recognize specific sequences in DNA for seq-sepcific binding
-basis of structural classification
-dimerization domain
activation domain (Q1 and Q2)
-interact with general transcription factors, RNA polymerase II, or other regulators of transcription
-ex: acidic domains, glutamine-rich domains, proline-rich domains
Protein Interaction domain (KID)
associate with proteins like histone acetyltransferases or coactivators
CREB order of domains
Q1 -> KID -> Q2 -> bZIP
TATA binding protein (TBP)
-uses large B-sheet surface to recognize DNA by binding in MINOR groove (still sequence-specific)
-recognizes TATA and other variations
-control which gene gets transcribed
-binding to TATA box creates mark for transcription to start
-binding induces change in DNA structure
TBP binding to TATA box
-induces significant change to DNA structure
-induced fit confers with sequence specificity = TBP bends the TATA box
-directs assembly of initiation complex by ordered addition of several general transcription factors and RNA polymerase II
TBP B-scaffold with minor groove contacts
-Lys and Arg interact with phosphate groups
-Phe groups jam into minor groove and kink the DNA
-binding specificity results from an induced-fit being energetically possible for TATA box sequences
TATA sequence flexibility
Steroid Nuclear receptors
-TFs regulated by steroid hormones
-initiate signalling processes activated by steroid hormones (estrogen, glucocorticoid, progesterone, androgen)
-receptor binds steroid then to specific site on DNA and activates transcription
-many associated with disease
DO NOT WORRY ABOUT
SEROTONIN RECEPTORS
Nuclear receptors
-DIRECT interaction with DNA and control of gene expression (unique property differentiates them from other receptors)
-key to embryonic development, adult homeostasis and metabolism
Steroid nuclear receptors associated with disease
-targets of 10-20% FDA approved drugs
-eg estrogen, androgen, progesterone, thyroid receptors
DBD of heterodimer nuclear receptor
binds DNA promoter regions
LBD of nuclear receptor
has agonist ligands and pepride from NR coactivator
Heterodimer nuclear receptor structure
LBD hing DBD
Steroid Hormone Nuclear Receptor Mechanism
- Bind hormone = conformational shift
- ligand-bound dimerized receptor recognizes HREs
- DBDs bind HRE
4.complex recruits transcription coactivators
hormone response elements (HREs)
-recognized by ligand bound dimerized receptor
-bound by two DBDs of the dimer
-highly specific
Binding of hormone to cytoplasmic receptor
-conformational change
-dissociation of heat shock proteins
-dimerization with another NR
-transloaction from cytoplasm to nucleus
