electrophoresis

technique used to seperate macromolecules by how they migrate through a gel subjected to an electric field

short strands make it through the fastest, larger strands lag

Electrophoretic Analysis of a protein purification

way to tell whether purification schemes are effective

displays proteins present at each step.

Homeodomain

DNA binding motif for a group of gene regulatory proteins important for development

contains three α-helices

2 and 3 form a HTH motif

3 is the recognition helix

lac operon

operon needed to digest lactose efficiently in E. Coli and some other enteric bacteria

can use lactose as form of energy but needs to make enzyme β-galactosidase to digest it into glucose

it is inefficient to prod enzyme when there is no lactose or when there is glucose present

two part control mech to ensure it expends energy only when nec

lac rep halts production in absense of lactose

cap assists in prod in absense of glucose

lac repressor

tetramer

when lactose is absent, lac repressor binds near to operator blocking lac operon promotor

when lactose is present, one of its metabolites binds to the repressor causing it to change shape so it can no longer bind to the operator

also contains HTH motif

CAP

catabolite activiting protein

presence of CAMP inversely proportional to presence of glucose

when glucose low, a lot of CAMP binds to CAP which aids in binding RNAP to promotor of lac operon which increases production of enzyme B-galactosidase

how to identify DNA binding experimentally?

1. gel shift

2. DNAase I footprinting

3. chromotin immunoprecipitation

Gel retardation assay

(electrophoretic mobility shift assay)

DNA is neg charged

moves toward positive charge in electrically charged gel

proteins slow migration rate

main idea: elecrophoretic mobility of a DNA fragment is reduced when protein is bound to it

DNAase I footprinting

main idea: protein binding protects DNA from endonuclease cleavage

can ID exactly location of binding site

seq of prot binding region can be det by comparison with marker DNA frags of known length analyzed on same gel

nuclease

enzyme capable of cleaving the phospodiester bonds betweem nucleotides in nucleic acids

two types: endonuclease and exonuclease

chromatography

technique used in labs for seperation of mixtures

involves passing a mixture dissolved in mobile phase through a stationary stage which seperated the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases

Enhancer

can be anywhere in gene

it is a cis-regulatory sequence that binds trans-acting factors that promote transcription by interacting with mediator complex

indep of orientation and position

transcription factor

sequence specific DNA binding factor

defining feature is the DNA binding domain

general transcription factor

bind to promoter region

many involved in forming of preinitiation complex

mediator complex

co-activators

bridge enhancers to promoter complex

co-regulators

(co-activators and co-repressors)

interact with transcription factors

one mechanism of action: modify chromatin

don't bind DNA themselves

how to find enhancer sequences?

5' deletion analysis

use reporter constructs

also DNAase I footprinting

x ray crystallography

grow protein crystals

irradiate with Xray

crystal diffracts rays onto a detector (film)

forms xray diffraction pattern

can make an electron density map

bad for membrane proteins--they don't crystalize well--neither do partially folded polypeptide chains

protein purification

proteins can be purified according to:

1. solubility

2. size

3. charge

4. binding affinity

way to purify proteins according to size

dialysis

gel filtration chromotography

Gel filtration chromatography

underlying principle: particles of diff sizes will filter through at different rates

proteins seperated according to size

largest exit first

because large proteins cannot enter internal volume of beads, they emerge sooner than do small ones

Core Promoter Elements

either TATA box

OR

Initiation element (INR) and Downstream Promoting Element (DPE)

Yeast enhancer

Upstream Activating Sequence UAS

usually only one per yeast gene

Drosophila Segment Cascade

maternal (bcd)

gap (hb)

pair rule (eve/ftz)

segment polarity (en)

NtrC

transcription factor

activator

mechanism: looping

opens DNA for polymerase to enter and start transcribing; gets Pol going

Sp1

transcription factor

activator

binds proximal promoter sequence and TAFs--helps build promoter complex

CtBP

co-repressor

repressor recruits this protein to DNA

which recruits HDACs

mechanism: local repression by deacytylation (chromatin remodelling)

HDACs

takes acetyl groups off histones

chromatin remodelling factor

both short range and long range

recruited by CtBP (short) and Groucho (long)

example of cooperative binding

(activators)

NFAT and AP1

two activating transcription factors

co-transfection assay

way to tell if protein directly regulates expression of a certain gene

add phage that makes protein of interest

add phage with reporter construct (binding sites for protein and selectable marker gene  such as lacZ)