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