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Biochem > Lecture 9: DNA-binding Proteins > Flashcards

Lecture 9: DNA-binding Proteins Flashcards

1
Q

Sequence-specific DNA-protein recognition and DNA-binding proteins

A

-certain sequences bind with higher affinity than others
-recognize base-specific interactions
-altered DNA structure
-involves binding major groove

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2
Q

Base-specific interactions

A

H donor, acceptor, nonpolar groups of bases

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3
Q

DNA-binding proteins and nonspecific interactions

A

-recognize interactions of DNA phosphate backbones and sugars
-involves binding minor groove

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4
Q

DNA-protein complex involving induced fit-binding

A

-can have sequence specificity
-when binding alters DNA structure so that it is not B-form
-favoribility of induced fit depends on sequence

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5
Q

DNA-binding proteins

A

-histones
-transcription factors
-polymerases and nucleases

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6
Q

Nucleosome

A

-unit of chromatin (beads on a string)
-146 bp DNA in 1.65 turns wound around histone octamer

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7
Q

Histone octamer

A

-DNA wraps almost twice around core = 2x the natural curvature of B-form DNA
-2 copies of each H2A, H2B, H3, H4

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8
Q

Energy required to deform DNA

A

from electrostatic interactions between DNA and histones

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9
Q

Linker DNA of 20-90bp

A

occurs between histones

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10
Q

Histone side chains

A

-interact with DNA-sugar-phosphate backbone
-ARG/LYS residues insert into minor groove
-contact areas are spaced to bend the DNA around histone core

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11
Q

N-terminal tails of histones

A

-protrude from core
-become sites for acetylation to regulate gene expression

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12
Q

Histone deacetylases (HDAC)

A

-cancer targets
-inhibited by SAHA

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13
Q

SAHA

A

-first FDA approved HDAC inhibitor
-clinically effective treatment of some cancers

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14
Q

DNA coiling around histone core

A

-negative supercoil created from one wrap around histone
-compensating positive supercoil forms outside of core then is relaxed by topoisomerase
=one (net) negative supercoil

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15
Q

Lk in DNA wrapping

A

Lk is constant because ends are fixed as if circular

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16
Q

DNA sequence dependence for location of nucleosomes

A

-some exist based on energetic preferences for the bending required to form the nucleosome particle

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17
Q

helix-turn-helix (HTH) motif

A

-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

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18
Q

Zinc Fingers (ZnFs)

A

-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

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19
Q

ZnF amino acid domain

A

-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

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20
Q

ZnF modules

A

-structurally diverse: 8 fold groups
-present in 1000 different protein

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21
Q

Basic region-leucine zippers (bZIP)

A

-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

22
Q

bZIP examples

A

-fos and jun
-cAMP response element-binding protein (CREB)

23
Q

Helix-loop-helix (HLH) motif

A

-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

24
Q

HLH transcription factors

A

-myoD
-myc
-max

25
Q

Transcription factors

A

-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

26
Q

Transcription factor-DNA interactions

A

-common interactions between dimeric proteins and palindromic sequences
-binding often leads to conformational changes in protein and DNA

27
Q

Transcription

A

-use DNA to make RNA
-highly regulated
-off=DNA. condensed into nucleosomes
-on=transcription factors activating transcription

28
Q

Regulation by transcription factors

A

-bind either promoter or enhancer regions of DNA
-recruit coactivator/corepressor proteins to the complex
-stabilize/block RNA polymerase
-catalyze de/acetylation of histones

29
Q

Transcription factor function

A

-basal level transcription
-development
-signaling
-cell cycle control

30
Q

Regulation of transcription factors

A

-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)

31
Q

Eukaryotic TFs

A

-modular structure (multiple domains)
-ex: Creb

32
Q

CREB

A

-DNA binding domain (bZIP)
-activation domain (Q1 and Q2)
-Protein interaction domain (KID)

33
Q

DNA binding domain (bZIP)

A

-recognize specific sequences in DNA for seq-sepcific binding
-basis of structural classification
-dimerization domain

34
Q

activation domain (Q1 and Q2)

A

-interact with general transcription factors, RNA polymerase II, or other regulators of transcription
-ex: acidic domains, glutamine-rich domains, proline-rich domains

35
Q

Protein Interaction domain (KID)

A

associate with proteins like histone acetyltransferases or coactivators

36
Q

CREB order of domains

A

Q1 -> KID -> Q2 -> bZIP

37
Q

TATA binding protein (TBP)

A

-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

38
Q

TBP binding to TATA box

A

-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

39
Q

TBP B-scaffold with minor groove contacts

A

-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

40
Q

TATA sequence flexibility

A
41
Q

Steroid Nuclear receptors

A

-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

42
Q

DO NOT WORRY ABOUT

A

SEROTONIN RECEPTORS

43
Q

Nuclear receptors

A

-DIRECT interaction with DNA and control of gene expression (unique property differentiates them from other receptors)
-key to embryonic development, adult homeostasis and metabolism

44
Q

Steroid nuclear receptors associated with disease

A

-targets of 10-20% FDA approved drugs
-eg estrogen, androgen, progesterone, thyroid receptors

45
Q

DBD of heterodimer nuclear receptor

A

binds DNA promoter regions

46
Q

LBD of nuclear receptor

A

has agonist ligands and pepride from NR coactivator

47
Q

Heterodimer nuclear receptor structure

A

LBD hing DBD

48
Q

Steroid Hormone Nuclear Receptor Mechanism

A
  1. Bind hormone = conformational shift
  2. ligand-bound dimerized receptor recognizes HREs
  3. DBDs bind HRE
    4.complex recruits transcription coactivators
49
Q

hormone response elements (HREs)

A

-recognized by ligand bound dimerized receptor
-bound by two DBDs of the dimer
-highly specific

50
Q

Binding of hormone to cytoplasmic receptor

A

-conformational change
-dissociation of heat shock proteins
-dimerization with another NR
-transloaction from cytoplasm to nucleus

Biochem (30 decks)

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