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Same nuclear genome means
a differentiated cell contains all the genetic instructions
necessary to direct the formation of a complete organism
- if take a differentiated cell from a frog and remove its nucleus and inject it into an unfertilized egg with uv light destroyed nucleus, a normal embryo will form and a tadpole will be developed
- Protein 2- dimensional gel- electrophoresis
- Proteins separated by charge in the first dimension and
by molecular weight in the second dimension
- in the example given the human liver and brain tissue have the same dna w/ diff expression patterns leading to diff protein composition
- bc most rna are not ready to be used, so rna processing is required.
- there are many ways to interupt this process to control gene expression
Six steps at which eukaryotic gene expression can be controlled
1. transcriptonal control
2. rna processing control
3. rna transport & localization control
4. translation control leads to protein
5. mRNA degradation control leads to inactive mRNA
6. protein activity control
bind to the
surface of DNA without
opening the double-helix.
components of genetic
- loop helix
- turn helix
- leucine sipper
and most common DNA-binding motifs.
- 2 helix connected by trun structure
Important role in
sequences in DNA
- 2nd helix stabilizes recognition helix, inturn stabilizing interaction with dna
- lamda repressor
- tryptophan repressor
- they have different pathways in common.
-distance is always the same (3.4). Formed as a dimer, but distinct 3.4 bc of major groove
mediates dimerization and DNA binding
- 1 stucture used for dimerization
- 1 struc used for interacting with dna
- in an active HLH homodimer both struc are present. In a inactiave heterodimer dna binding struc is missing & dimerization alone cannot activate transcription factor
- mediates both dna binding and dimerization:
Two alpha helical DNA binding
domains dimerize through
their alpha helical leucine zipper region to form an inverted Y-shaped
by a single
alpha helix, one from each monomer,
that mediates binding to a specific DNA
sequence in the major groove of DNA
Gcn4 protein: transcription factor
regulating amino acid biosynthesis
- results in different sequence
- detects sequence-specific
- DNA is negatively charged and
migrates to the positive electrode.
- Free DNA migrates fast through the
- DNA covered with proteins will
- purified protien that binds to dna frag is used.
- add nuclease to get random cleavage getting varies peices of dna frag
- adding specific protein to dna frag will protect frag from digestion getting blank portion in gel electrophoresis where no cleavage is observed
Total cell proteins
lo salt wash to remove non binding proteins
med salt wash elutes many diff binding proteins
Dna binding proteins
med salt wash removes proteins not specific for certain dna hi salt wash elutes specifc dna binding proteins
Switching the tryptophan genes on and off:
- if the end product is present it will activate repressor protein preventing transcription.
- The end-product tryptophan binds to the repressor.
The repressor protein binds then to DNA and prevents transcription
Summary of the mechanisms by which specific gene
regulatory proteins control gene transcription in prokaryotes:
A) negative regulation: bound repressor protein prevents transcription
B) positive regulation: bound activator protein promotes transcription
- glucose will always be the 1st choice of carbon source.
- so if glucose is present, lactose will not be used and operon will be turned off
- it is found in the gene control region of eukaryotic cells.
- helps to establish interactions
between the components to activate trancription
Binds general transcription factor
TFIID which is needed for the
recruitment of RNA polymerase II
to the promoter
- 1 activator at a specific location give off 1 unit of transcription
- 1 activtaor at another specific location gives off 2 units of transcription
- both activators together gives off 100 units of transcription
Eukaryotic gene regulatory proteins often
assemble into complexes on DNA:
- DNA looping can contribute to gene activation at a distance.
- it causes efficient dna transcription
- as long as the dna binding domain on the activator protein and the recognition seq on the dna are specific to each other, it doesnt matter what activation domain it has
1. protein synthesis (only when needed)
2. ligand binding
3. covalent modification (phos)
4. addition of 2nd subunit (dna binding subunit +activator subunit)
5. unmasking ( phos)
6. stim of nuclear entry
7. release from membrane (binds to dna)
A single gene regulatory protein can coordinate do what?
- can coordinate the expression of several different genes
- 1 transcription factor when activated can activate mult genes in 1 pathway in response to 1 ligand.
ex) glucocorticoid hormone
Eukaryotic activator proteins can do what?
alter local features in
chromatin structure to stimulate transcription initiation
- dna is wrapped by histones in nucleosomes forming (+) charged chromatin. the (+) charge will tightly interact with (-) phos of dna backbone.
- dna has to be opened to initiate trancsription
- chromatin remodeling complex
- histone modifying enzyme
By moving nucleosomes, proteins like transcription factors can get access to DNA that was previously unavailable, wrapped around nucleosome cores
1) remodeled nucleosomes
- histone chaperone
2) histone removal
- acetylation (+)
- By remodeling the chromatin and
modifying histones, chromatin will open
- Transcription factors can bind to the
promoter region and activate the gene.
1. competitive dna bindin
2. masking the activation surface
3. direct interaction with the general transcription factors
4. recruitment of chromatin remodeling complexes
5. recruit of histone deactylases
5. recruit of histone methyl tranferases
- prevents histone modification
- removes acetyl group forming a compact chromatin structure by allowing (+) charge of histones to reappear.
Ex of deacetylase) HDAC
- in dna gene A and gene B are both controlled by the same gene regulatory seq.
- the enhancer should go both genes, but since gene A should be turned off, so the insulator element prevents the enhancer from activating it
- separates heterochromatin from the rest of the chromatin.
- will not let the heterochromatin spread to active chromatin.
- if barrier is not present heterochromatin will spread inactivating the entire chromatin
Formation of 5-methylcytosine
methylation of a cytosine
base in the DNA double helix.- when dna replication occurs methylation stays with the original strand and then it is translated to the replicated strand.
Mechanisms that can
produce a form of epigenitic
inheritance in an organism
- epigenetics is the study of heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence
- positive feedback
- histone modification
Schematic diagram showing how a positive
feedback loop can create cell memor
- protein A in a cell is not made bc it is normally required for its own trancription
- a transient signal turns on expression of protein A
- the effect of the transient signal is remembered in all the cell descendants
- it inactivates chromatin and the new inactivated chromatin state is inherited.
- 1 strand will ahve histone modification but new strand has to inherit it
methylates a dna region and the new dna methylated state is inherited
- new dna will have histone modification
- used for gene silencing
- dna methylation: The addition of methyl groups to the DNA changes its binding properties, so that it no longer binds to this machinery, and that copy of the gene is not transcribed
- females have 2 X chromosomes and 1 must be silenced by chromosome condensation.
- the end result is a tighly packed X chromosome which is inactivated
- males only have 1 X chromosome so they are always expressed
different modifications to inhibit RNA transcription so you cannot form a protein
-RNA trancript aborts
-nonfunc mRNA seq
-retention & degradation in nuclues
- RNA degraded
- allows regulation of gene transcription
- the riboswitch controls expression of the purine biosynthetic genes.
- when guanine levels in cell are lo an elongating RNA polymerase transcribes
the purine biosynthetic genes.
- if guanine is abundant it will bind to riboswitch causing shape change that makes RNA polymerase stop transcription
-mutually exclusive exons
-internal splice site
- Specific nucleotides are inserted into the mRNA sequence or existing
nucleotides are changed by deamination (e.g. adenine to inosine, cytosine to uracil).
- Results: alteration in protein sequence.
Mechanisms for the
RNA are not evenually ditributed
- directed transport on cytoskeleton
- random diffusion and trapping
- generalized degradation in combination with local protection by trapping
Mechanisms of translation control
1. translation repressor protein
2. increased temp
3. small molecule (end product) maskes it
4. antisense rna: micro rna causes degradation of specific rna
- Short non-coding RNAs.
- Humans express more than 400 different miRNAs, and these appear to regulate at least one-third of all human protein-encoding genes.
Once made, miRNA base-pair with specific mRNA and regulate their stability & translation. The miRNA undergo a special type of processing, after
which the miRNA is assembled with a set of proteins to form an RNA-induced
silencing complex (RISC)
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