Regulation Of Gene Expression

In Eukaryotes
Features
Amount of genetic
information
Chromosome organization

Prokaryotes

Eukaryotes
Much more

Some proteins

Nucleus and other

compartments
Number of chromosomes
Transcription and translation

none

Complex of DNA, histones,

proteins
Double-membrane bound

mRNA transcripts

Not processed

Number of cells, tissues

unicellular

one
No separation

Many linear
Spatially and temporally
separated
Processed before transport
to cytoplasm, mRNA more
stable
multicellular

Cloning of a frog from a skin cell

Elegant experiments in Xenopus
laevis by John Gurdon, following
earlier experiments in
Rana temporaria by Briggs and
King (1952), provided strong
evidence that the genetic content
of differentiated somatic cells is
essentially unchanged from that
of the early embryo.

Dolly The Sheep

Regulation Of Gene Expression

In Eukaryotes
Regulation in the nucleus
Chromatin organization level chromatin
remodeling
DNA level controlled transcription
RNA level alternate splicing

Regulation in cytoplasm
RNA level stability
Polypeptide level - compartmentalization

Regulation On Transcriptional
Level
Whether a particular gene can be
transcribed and how often

Regulation On Transcriptional
Level
Organization of DNA in the nucleus

Gene Activity Regulation at the

Chromosomal Organization Level
Structure of chromatin is the major switch on/off
Transcriptionally active genes on the chromosome
are cycled to the edge of the chromosome territory

Gene Activity Regulation at the

Chromosomal Organization Level
Nucleus highly organized
A chromosome territory
gene dense chromosomes more internal;
interchromosomal compartments
Rearrangements of chromosomal structure

Regulation On Transcriptional
Level
Chromatin Remodeling

Regulation at Chromatin
Remodeling Level
Heterochromatin transcriptionally inactive
Euchromatin sensitive to DNase I digestion,
transcriptionally active
Chromatin remodeling:
Change in interaction between DNA and histones
Remodeling done by diverse group of proteins with
ATPase activity
After remodeling, promoters become accessible for
proteins

Histone Code
Acetylation
Methylation
Phosphorylation

The Histone Code

Methylation of histone (or of DNA)
usually turns a gene off
Acetylation of histone usually turns a
gene on
Phosphorylation -- we're not sure what
that does.

Histone Code

Chromatin Remodeling
Methyl groups can be added to lysine and
arginine gene inactivation
Phosphate groups added to hydroxyl groups
of serine and histidine introducing a
negative charge chromatin unpackaging
during replication

Nucleosome Altering
Nucleosome altering can be done by:
Altering the contacts by DNA and histones
Moving DNA off nucleosomes
Nucleosome core particle altered, producing a
nucleosome dimer

Chromatin Remodeling
Best studied complex of proteins that
remodel chromatin SWI/SNF:
From yeast to humans
11 subunits complex
One subunit has a domain for non-specific
DNA binding
Second subunit is ATPase

Chromatin Remodeling

Histone Modification
Another mechanism of chromatin alteration
Catalyzed by histone acetyl transferase
(HAT)
acetate group added to basic amino acids on
histone tail
Attraction between basic amino acids and acidic
DNA is lessened
Remodeling moves from promoter to
transcription unit (coding region)

HAT/HDAC
HATs are targeted to genes by specific
activator proteins
Can be reverted histone deacetylase HDAC
co-repressors, remove acetyl groups
Insulator elements short DNA sequences
that bind specific proteins and act as barriers
that prevent the spread of remodeling into
neighboring genes

A model for transcriptional

repression

Regulation On transcriptional
Level
Transcriptional Factors

Regulation of Transcription:
Proteins Involved
Proteins involved in control of transcription
transcription factors, TFs
Contain two domains
DNA binding domain
Protein binding domain protein interaction

Regulation of Transcription:
Proteins Involved
TFs bind at enhancer sites
Positive factors, activators high
transcriptional levels
Negative factors, repressors low
transcriptional level

DNA Binding Domains

DNA binding domain characteristic 3-D
patterns or motifs:
Helix-turn-helix
Zinc finger
Basic leucine zipper (bZIP)

Structural motifs within different

types of transcription factors

DNA Binding Domain Motifs

Trans-activating Domain
Transcription factors also contain a transactivating domains that interact with basal
transcriptional complex
Different from DNA-binding domain
Interact with other TFs or directly with RNAPs

Other domains bind small proteins

hormones, small metabolites

Regulation On Transcriptional
Level
DNA sites involved in regulation of
transcription

Regulation of Transcription:
DNA Sequences Involved
cis-acting regulatory sequences
Promoters
Enhancers
Silencers

Promoters

Region necessary to initiate transcription

TATA, CAAT, GC boxes
TATA box core promoter

region of RNA polymerase binding,

-25-35 bp upstream,
7-8 bp consensus sequence,
(AT)n flanked by GC

CAAT box consensus CAAT or CCAATT,

-70-80 bp upstream
region of regulatory proteins binding (RPBS)

GC box consensus GGGCGG,

-110 upstream
bind transcription factors

Alignment of multiple
promoters

Strong/Weak Promoters
The more similarity to a consensus
TATAAT, the stronger promoter
Less similarity lower level of transcription
Up mutation makes it stronger
Down mutation makes it weaker

Identifying promoter sequences

required for transcription

Enhancers
Similar to operator in prokaryotes, in eukaryotes:

Act over relatively large distance remote control

Position not fixed can be upstream, downstream
Orientation can be inverted
Unspecific enhancement?

Difference from promoters:

Promoters give basal level of transcription
Enhancers provide full level transcription
Responsible for time and tissue specificity

Enhancers
Enhancer within gene:
Immunoglobulin heavy chain, in an intron
between two exons

Enhancer downstream
Beta-globin gene in humans
Thymidine kinase in chicken

Enhanceosome
Activators A + E = enhanceosome
Gene-specific transcriptional switches

Transcriptional activators bound

at distant sites can influence gene
expression

Regulation of Transcription:
TF in Transcription Initiation
Transcription complex assembly on
promoter
Transcription factors are different for
different RNA polymerases
RNA polymerases
RNAP I r RNA
RNAP II mRNA, snRNAs
RNAP III tRNA, 5SRNA

RNA Polymerase II
For RNA polymerase to bind to a promoter
general transcription factors (TF) assemble
at promoter in a specific order
General TFs for
____________________________________
Form PIC pre-initiation complex
Provide a platform for RNAP II to recognize
___________________________________

PIC
TFIID binds to TATA box through TBP (TATA
binding protein) + 13 TAFs (TATA associated
factors) 20 bp of DNA bind TFIID
TFIID binds to an activator TFIIB
TFIIB interacts with TBP and DNA sequence
upstream of TATA box
Then TFIIA, RNAP II, TFIIF, E, H, J
RNAP II leaves TATA box
Transcription on basal level

PIC

Silencers
Gene silencing transcriptionally silent
Silencers DNA sequences that mediate
this process
Specific protein complex that prevent
binding with transcriptional enzymes
Positional effect
RNAi ____________________________

Regulation At DNA Replication

Level- Methylation
DNA methylation
DNA modification by methylation of phosphates
and sugars
Base methylation CH3 to cytosine
2-7% of Cs are methylated
Extent of methylation is tissue specific
Degree of methylation = degree of expression
Methylation patterns are tissue specific and
heritable

DNA Methylation
40% of base pairs are G-C
5-mCpG 3
3-GpCm-5 mCpG is not digested by methyl sensitive
endonucleases

CpG islands 1-2 kb long, 30,000 in human

genome, near transcription sites
No methylation of Cs
DNA is hypersensitive to DNaseI
Contain less H1 histone

Expression of the XIST gene in

the inactive X chromosome of
human females

DNA Methylation and Imprinting

Methylated DNA is transcriptionally repressed
Human X-chromosome is extensively methylated
Two proteins known to bind to methylated DNA

Methylation is controlled by parental origin

imprinting
If methylated in mother, only allele of mothers
chromosome will be methylated

Processing Level Control

Determine the path by which premRNA is processed to mature mRNA

Processing Level Control

Alternative Splicing

Alternative Splicing

Regulation at mRNA Level

Alternative Splicing
Alternative splicing produces many different
________________________________________.
Examples:
Tissue specific gene expression:
Calcitonin or CGRP (Calcitonin Gene Related Peptide)
(hormones) in the calcitonin gene transcript from rats
depending on tissue type
Alpha-tropomyosin

Tropomyosin

Calcitonin

Sex In Drosophila

Translational Level Control

Determine whether a particular
mRNA is actually translated and how
often, for how long

Translational Level Control

Localization of mRNAs to certain sites
within a cell
Whether or not an mRNA is translated and
how often
The half-life of mRNA

Translational Level Control

UTRs contain nucleotide sequences used by
the cell to mediate translational level control
Cytoplasmic localization of mRNA is
governed by 3 UTR zip codes
Motor proteins recognize them as tracks,
transport mRNA to particular location
Microfilaments anchor mRNAs

Cytoplasmic Control Of mRNA

Stability
mRNA ribosomessynthesis degradation
(control point)
mRNA longevity

Poly(A) tail stabilizes mRNA

Sequence of 3UTR AUUUA are short lived
Chemical factors hormones
siRNAs, miRNAs lead to mRNA degradation

mRNA degradation in
mammalian cells

RNA Interference
Gene silencing by degrading mRNA
through RNAi mechanism