1. Eukaryotic Gene Regulation
Last lecture we covered elements of the eukaryotic gene and
described how each contributed to the creation of a functional
mRNA, capable of producing a protein
Basal level transcription = negligible physiological effect
Induced level transcription = caused by transcription factors
The elements of the gene covered last lecture are part of the
network of gene expression control points

2. Review: Central Dogma Revised
DNA
RNA
PROTEIN
1 GENE
1 ENZYME
This is an oversimplification
Multiple genes can arise from the same DNA
Some genes encode subunits of large enzyme complexes
(ex: RNA polymerase)
Genes can have multiple functions (sometimes tissue-based)
Catalytic RNAs, tRNAs, rRNAs (not proteins)
Genes can have multiple reading frames (six possible)

3. Genes Within Genes Three reading frames per strand
Possible to have promoters at
either end of a gene (ie, both
strands are template strands)
Ex: Mitochondrial ATPase genes
Molecular Biology of the Cell

4. Control Points for Eukaryotic Genes
cytoplasm
nucleus
DNA
I. Chromatin Structure &
Chemical Modification
RNA
II. Transcription
mRNA
III. RNA Processing
IV. Nuclear Export
V. Translation
Protein
VI. Protein Modification
& Trafficking

5. Control Points for Eukaryotic Genes
cytoplasm
nucleus
I. Chromatin Structure &
Chemical Modification
DNA
RNA
mRNA
Protein

6. What causes these areas to be condensed?
Review: Euchromatin & Heterochromatin
The structure of chromatin gives an opportunity to control
gene expression
Heterochromatin = highly condensed, unable to transcribe
Euchromatin = relaxed, areas of transcription
What causes these areas to be condensed?

7. Histone Modification & Gene Expression
Remember: Histones bind to DNA because the many (+)
charged amino acids (Lys & Arg) bind to (-) PO4 groups of DNA
Transferring an acetyl group (COCH3) to these amino acids
nullifies their (+) charge
Without this electrostatic interaction, DNA “falls” off the
nucleosome
The N-terminal tails of H3 and H4 are the most common sites of
acetylation
Heterochromatin is less acetylated than euchromatin, which
causes its more condensed conformation

8. Histone Modification & Gene Expression
Histone acetyl transferases
(HATs) are the enzymes that
acetylate histone proteins
Many transcription factors
have HAT activity; this is
how they unwind the
nucleosome structures of
promoters
Transcriptional repressors
can recruit histone
deacetylases (HDACs) to
promoter sites to condense
the DNA

9. DNA Methylation
Methylation is another way of controlling gene expression
Histone proteins can be methylated as well as acetylated
DNA can be methylated directly, mainly at C residues in CG
dinucleotide pairs
Methylated DNA is transcriptionally inactive
Factors like Methyl-CpG binding protein will bind to methylated
sites and have HDAC activity
Heterochromatin is highly methylated

10. Control Points for Eukaryotic Genes
cytoplasm
nucleus
I. Chromatin Structure &
Chemical Modification
DNA
II. Transcription
RNA
mRNA
Protein

11. Transcription Initiation Control
Primary site of gene expression control
We know that basal level transcription is achieved by the basal
initiation complex of general TFs
Gene-specific TFs accelerate transcription to significant levels
These TFs can bind to promoter elements upstream of the RNA
polymerase binding site (TATA box) or to enhancers

12. Transcription Factor Structure
Transcription factors usually have two major domains:
DNA-Binding Domain  binds TF to consensus sequence of the promoter
Activation Domain  binds TF to consensus sequence of the promoter
These two domains are modular; ie they can be switched and
still function
Called “domain swapping”
More on this when we talk about yeast

13. Domain Swapping Experiments
GAL4 PROMOTER
REPORTER GENE
Molecular Biology of the Cell
GAL4 binds to its promoter normally
Combine GAL4 activation domain with LexA binding domain
Now it won’t bind to GAL4 Promoter
But it will bind to LexA promoter and activate the gene

14. Transcription Factor Binding
DNA-binding domains of TFs attach to consensus sequences by
hydrogen bonds
This binding usually occurs on bases exposed in the major
groove; minor groove is usually too tight for domains to fit
Leninger Principles of Biochemistry

15. DNA-Binding Domains: Helix-Turn-Helix
HTH domain is also
called homeodomain,
because it is found in
homoeobox (hox) genes
Conserved amino acids
of recognition helix bind
to major groove bases
Example: lac repressor

16. DNA-Binding Domains: Leucine Zippers
Contain 4-5 Leu residues along
one face of a helix
Hydrophobic interactions of the
Leu residues cause this factor to
form a homodimer or heterodimer
The adjacent helix on each
monomer has many (+) charged
residues
These helices bind DNA in the
major groove in a “scissor grip”
Ex: c-fos & c-jun (cell cycle
TFs, form heterodimers)

17. DNA-Binding Domains: Zinc Finger
Conserved pairs of Cys & His
residues bind a Zn atom
A stem-loop structure (the finger)
binds to DNA in the major groove
These “finger” motifs usually
occur in groups of three
Sometimes the two His residues
will be Cys (ie, four Cys total)
Ex: TFIIIA

18. DNA-Binding Domains: Basic Helix-Loop-Helix
Similar to Leu zipper
Bind as homo- or hetero-dimers
Basic, (+) charged DNA binding
domains, bind in “scissor grip”
Two pairs of helices join through
hydrophobic interactions
Ex: c-myc, a cell cycle gene

19. DNA Activation Domains
Much more varied in structure than DNA-binding domains
Act in a variety of ways to produce one of two specific effects:
I. Alter the physical structure of the promoter to aid General TF Binding:
Recruit HATs
Unwind DNA
II. Increase the binding efficiency of, or stabilize General TFs
Bind to General TFs and induce conformational changes
Activate General TFs by chemical modification

20. Control of Transcription Factors
Human molecular genetics
c-myc, c-fos, c-jun
Cell Division
What induces a
transcription factor to
bind to DNA?
Signaling Pathways
A hormone or other factor binds to a
receptor on the cell surface
Signal transduction pathways activate,
the ultimate result of which is the
recruitment of a transcription factor
Signaling can initiate expression of a
transcription factor or activate (ex:
phosphorylate) a factor that has
already been transcribed

21. DNAse Footprinting Method of detecting transcription factor binding
DNaseI cannot digest DNA that is
bound with protein
Electrophoresis of digested DNA
will reveal the region bound by a
transcription factor

22. Control Points for Eukaryotic Genes
cytoplasm
nucleus
I. Chromatin Structure &
Chemical Modification
DNA
II. Transcription
RNA
III. RNA Processing
mRNA
Protein

23. Alternative Splicing mRNA is capped, poly-adenylated and spliced
Splicing can occur in different patterns, thus producing totally
different gene products
Since exons often represent protein
domains, excluding an exon by splicing
results in a protein with different
function (ex: localization signal)
Trans-splicing can occur; exchanges
exons between different mRNAs
Ex: trypanosomes

24. Drosophila sxl: Sex Determination by Splicing
Stop codon present in this exon
Stop codon present in this exon

25. Alternative Splicing for Tissue Specific Expression
mRNA can be spliced to create gene products with different
functions or different sites of expression.
Remember that some introns contain genes
Why?
 efficiency

26. Control Points for Eukaryotic Genes
cytoplasm
nucleus
I. Chromatin Structure &
Chemical Modification
DNA
II. Transcription
RNA
III. RNA Processing
mRNA
IV. Nuclear Export
Protein

27. mRNA Export
mRNA must be translocated from the nucleus to the cytoplasm
for translation
mRNA is inherently unstable; easily degrades (mRNA decay)
Capping and poly-A tract aids stability and against nucleases
mRNA is not usually linear; forms secondary structures that help
keep it stable
Secondary folding also helps compact it for travel through the
nuclear pore complex

28. mRNA Secondary Structure
3-Stem Junction
Hairpin
Bulge
Stem-Loop
Symmetric
Internal Loop
Asymmetric
Loop

29. Control Points for Eukaryotic Genes
cytoplasm
nucleus
I. Chromatin Structure &
Chemical Modification
DNA
II. Transcription
RNA
III. RNA Processing
mRNA
IV. Nuclear Export
V. Translation
Protein

30. Binding of IRE-BP prevents ribosome complex from assembling
Translational Control of Gene Expression
Control of translation usually occurs at translation initiation
Ribosome complex must form on the mRNA template in order
for translation of the protein to occur
This form of control is less efficient (mRNA already produced)
Ex: Iron Response Element Binding Protein – blocks ferritin
translation when Fe is absent
Binding of IRE-BP prevents ribosome complex from assembling

31. Control Points for Eukaryotic Genes
cytoplasm
nucleus
I. Chromatin Structure &
Chemical Modification
DNA
II. Transcription
RNA
III. RNA Processing
mRNA
IV. Nuclear Export
V. Translation
Protein
VI. Protein Modification
& Trafficking

32. Protein Modification and Trafficking
After translation, proteins are folded & chemically modified
One polypeptide can have multiple functions if processed
different (again, 1 gene ≠ 1 enzyme)
Protein modification occurs in the Golgi apparatus
Gene expression is not complete until the protein reaches the
region of the cell where it is useful
Protein trafficking generally occurs via vesicles that bud off
the Golgi apparatus and direct the protein to an organelle
The destination of a protein is determined by its sequence
and modifications and can be controlled to accommodate the
cell’s metabolic needs

33. Post-Translation Modifications
PURPOSE
Peptide bond cleavage
Expose an active site
(ex: zymogens)
Glycosylation
Variable
(ex: cell-surface antigens)
Addition of Lipids
Membrane-surface proteins primarily
Enhance Structure & Stability
Disulfide Bond Formation
Hydroxylation
Structural (ex: collagen)
Activate Enzymes or TFs
Phosphorylation