9: DNA binding proteins
How do proteins bind to non-specific regions of DNA?
(low affinity) -Interact with DNA phosphate backbones and sugars -minor groove binding
Explain the Nucleosome and Topoisomerace mechanism
(sequence dependence exists for location) DNA wraps around histone core (negative supercoil) -> looks like once around histone and then an unbound positive supercoil forms (looks like an infinity sign) the unbound positive supercoil outside the core is compensate by Topoisomerase --> relaxes the DNA and leaves 1 negative supercoil (unbound big circle, no longer an infinity sign)
How do proteins bind to DNA?
- by recognizing a specific DNA sequence (high affinity) or - without any sequence preference (low affinity)
How do proteins bind to specific regions of DNA?
- specific interactions between DNA base pairs with the protein sidechains. - differences in the energetic cost to change the DNA B-form structure to the structure induced upon binding.
What stabilizes bZIP?
- through electrostatic interactions the basic amino acids interact with the negatively charged DNA backbone -in addition to forming H-bonds in the major groove
What do Zn fingers recognize?
-DNA... a-helix sequence specific along major groove via H bonds -RNA -other proteins
What are some base specific interactions recognized by DNA binding proteins?
-H-bond donor -acceptors -nonpolar groups of bases
What are HLH responsible for?
-are NOT responsible for DNA-binding (like Zn Fingers) -but for dimerization with another protein (like bZIP)
Nucleosome structure
-basic unit of chromatin -disk like structure -8 core histones → DNA wraps 1 ¾ around octamer (negative supercoil) -linker DNA between histones
What are some examples of DNA binding proteins?
-histones -transcription factors -polymerases and nucleases
What are some modular domain motifs?
1. DNA binding domain (bZIP) 2. activation domain (Q1 and Q2) 3. Protein interaction domain (KD)
What is the structure of a Helix-loop-helix?
2 amphipathic a-helixes connected by a loop 1 helix has positive charges and binds to major groove (DNA binding domain)
TATA-binding protein (TBP)
A general transcription factor that binds to the TATA box and assists in attracting other general transcription factors and RNA polymerase II to eukaryotic promoters.
What are zinc fingers?
A type of structure found in some DNA binding proteins. A zinc ion is complexed to 4 cysteines (or 2 Cys and 2 His per Zn) causing a loop of about 12 amino acids to protrude. An array of these occur in a repeating motif.--> tandem repeats Ultimate goal=therapeutics
What is a Nucleosome?
Bead-like structure in eukaryotic chromatin, composed of a short length of DNA wrapped around a core of histone proteins
a _____ involving induced fit can have sequence specificity
DNA protein complex
examples of HTH motif
E. coli lactose repressor homeobox domains: appear in developmentally important transcription factors
How do histones interact with DNA?
Histone side chain (+) interacts with DNA sugar phosphate backbone (-) Histone has Arg (+) and Lys (+) residues that insert into minor groove→ contact areas are spaced in a manner to bend DNA around octamer
What is the modular protein interaction domain?
KID associate with proteins like histone acetyltransferases or coactivators
How do histones play a role in gene regulation?
N-terminal tails of histones protrude form the core → sites for acetylation (chemical modification) → regulate gene expression By changing shape, the histones can expose different sections of DNA, called genes, at different times.→ The exposed genes determine what proteins will be made by the cell → gene expression (gene regulation).
what is the modular activation domain?
Q1 and Q2 Interact with: - general transcription factors - RNA polymerase II - other regulators of transcription (acidic domains, glutamine-rich domains)
The groups jam into DNA minor groove forming a kink during _____.
TATA binding protein binding to DNA
how many times does one nucleosome wrap around the core?
almost twice around the core→ generates high curvature (almost x2 as much as the natural curvature of B-form DNA)
What is the modular DNA binding domain?
bZIP -Sequence specific binding -Basis of structural classification -Dimerization domain→ promotes heter- or homodimer formation
What is bZip's binding domain composed of?
basic region arginine lysine
Where do electrostatic interactions occur?
between DNA and histones
what is a Basic region-leucine zipper (bZIP)?
contain leucine residues every 7th position along an alpha helix. A DNA binding domain is located 7 aa before the first leucine and alpha-helix
What are tandem repeats? How are they useful?
copies of genes repeated one after another along a chromosome. Ex: steroid hormone receptor transcription factors: use one Zn finger to bind to DNA and a second Zn finger to stabilize DNA binding by the first Zn finger
Topoisomerase
corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands
Transcription factor-DNA interactions: interaction between ____ and _____ → binding leads to conformational changes in protein and DNA
dimeric proteins palindromic sequences
What are electrostatic interactions?
energy required to deform DNA
nucleosome is a _____ for substantial bending
energy source
What stabilizes bent DNA bends substantially into a compact nucleosome?
histone electrostatic interactions
The leucines form a hydrophobic face that interact with the hydrophobic face of a similar alpha helix (protein-protein interaction) resulting in?
homodimer and heterodimer structure
What is an octamer?
is a group of core histones that help with packaging of nucleosomes 2 copies each of core histones form the octamer: (H2A, H2B, H3, H4)
Some DNA sequence dependence exists for ____________ of nucleosomes based on energetic preferences for the bending required to form the nucleosome particle
location
a _____ allows interactions with DNA and multiple other proteins.
modular structure (means multiple domains) Eukaryotic transcription factors have modular structure→ its multiple domains are responsible for various activities that are thereby grouped into motifs
Where do transcription factors bind?
promoter or enhancer regions of DNA
Helix-turn-helix (HTH)
proteins that regulate gene expression 20aa long formed by 2 helices connected by a short turn (3-5 residues long) 1st helix→ stabilizes motif 2nd helix→ recognition helix: binds in sequence specific manner in major groove
What are transcription factors?
sequence specific DNA-binding factor that controls the rate of transcription by promoting or blocking the recruitment of RNA polymerase
What is linker DNA?
the DNA (20-90bp) in between nucleosomes that is not wrapped around histones
energetic favorability/cost for induced fit depends on_______
the sequence
TATA binding protein binds by?
using large beta-shet to recognize DNA and bind in the minor groove sequence specific BUT minor groove binding