Gene Expression and

Regulation
I Made Winarsa Ruma, M.D., Ph.D.
• How does a gene, which consists of a string of
DNA hidden in a cell's nucleus, know when it
should express itself?
• How does this gene cause the production of a
string of amino acids called a protein?
• How do different types of cells know which
types of proteins they must manufacture?
DNA Transcription
• The genetic code is frequently referred to as a
"blueprint" for a cell to sustain itself.
• This code is the basis for the production of various
molecules, including RNA and protein.
• DNA is double-stranded, but only one strand serves as
a template for transcription at any given time
called the noncoding strand.
• The non-template strand is referred to as the coding
strand because its sequence will be the same as that
of the new RNA molecule.
 RNA polymerase (RNA pol)
• RNA pol attaches to the template DNA strand and begins to catalyze
production of complementary RNA.
• Composed of approximately a dozen subunits, and when active on
DNA, they are also typically complexed with other factors to
signal which gene is to be transcribed.
• Three different types of RNA polymerase exist in eukaryotic cell:
1) RNA pol I transcribes the genes that encode most of the
ribosomal RNAs (rRNAs),
2) RNA pol II that transcribes the messenger RNAs, which serve as
the templates for production of protein molecules.
3) RNA pol III transcribes the genes for one small rRNA, plus the
transfer RNAs that play a key role in the translation process,
as well as other small regulatory RNA molecules.
 Transcription Initiation
• The RNA pol binds to the DNA upstream (5′) of the gene
at a specialized sequence called a promoter.
• Eukaryotic promoters are more complex than their
prokaryotic.
• Many eukaryotic genes also possess enhancer
sequences.
• Enhancer sequences control gene activation by binding
with activator proteins and altering the 3-D structure
of the DNA to help "attract" RNA pol II, thus regulating
transcription.
• Because eukaryotic DNA is tightly packaged as chromatin,
transcription also requires a number of specialized
proteins that help make the template strand accessible.
• In eukaryotes, the "core" promoter for a gene
transcribed by pol II is most often found immediately
upstream (5′) of the start site of the gene.
• Most pol II genes have a TATA box (consensus sequence
TATTAA) 25 to 35 bases upstream of the initiation site,
which affects the transcription rate and determines
location of the start site.
• Eukaryotic RNA polymerases use a number of essential
cofactors (transcription factors), and TFIID recognizes the
TATA box and ensures that the correct start site is used.
• TFIIB, recognizes a different common consensus
sequence, G/C G/C G/C G C C C, approximately 38 to 32
bases upstream.
Eukaryotic core promoter region
• Enhancer act to enhance the rate at which genes are
transcribed, and their effects can be quite powerful.
• Contain multiple functional sequence elements
that bind different transcriptional regulatory proteins
• Enhancers can be thousands of nucleotides away
from the promoters with which they interact, but they are
brought into proximity by the looping of DNA.
• This looping is the result of interactions between the proteins
bound to the enhancer and those bound to the promoter.
• The proteins that facilitate this looping are called
activators, while those that inhibit it are called
repressors.
• Transcription of eukaryotic genes by polymerases I and III is
initiated in a similar manner, but the promoter sequences and
transcriptional activator proteins vary.
DNA looping
Action of
enhancers
Action of
eukaryotic
repressors
 Strand Elongation
• Once transcription is initiated, the DNA double helix
unwinds and RNA polymerase reads the template
strand, adding nucleotides to the 3′ end of the
growing chain.
• At a temperature of 37 degrees Celsius, new
nucleotides are added at an estimated rate of about
42-54 nucleotides per second in bacteria, while
eukaryotes proceed at a much slower pace of
approximately 22-25 nucleotides per second.
Transcription Termination
• In eukaryotes, termination of transcription occurs by
different processes, depending upon the exact
polymerase utilized.
• For pol I genes, transcription is stopped using a
termination factor, through a mechanism similar to
rho-dependent termination in bacteria.
• Transcription of pol III genes ends after transcribing a
termination sequence that includes a polyuracil
stretch, by a mechanism resembling rho-
independent prokaryotic termination.
• Termination of pol II transcripts, however, is more
complex.
Transcription Termination
• Transcription of pol II genes can continue for
hundreds or even thousands of nucleotides beyond
the end of a noncoding sequence.
• The RNA strand is then cleaved by a complex that
appears to associate with the polymerase.
• Cleavage seems to be coupled with termination of
transcription and occurs at a consensus sequence.
• Mature pol II mRNAs are polyadenylated at the 3′-
end, resulting in a poly(A) tail; this process follows
cleavage and is also coordinated with termination.
• Both polyadenylation and termination make use of
the same consensus sequence.
 Post-transcriptional steps in gene
expression
• All cells in the human body contain the same DNA
blueprint, with differential expression of those genes
confers distinct functional properties to each cell type.
• This differential gene expression is achieved at the level
of gene-specific transcription and numerous
post-transcriptional events that also contribute to
the cell-specific expression patterns, which dictate
function.
• Much of this post-transcriptional regulation of gene
expression occurs through the action of RNA binding
proteins and processing factors that associate with
RNAs from the initiation of transcription to the eventual
death of the RNA in the cytoplasm.
 Post-transcriptional steps in gene
expression
• Following transcription within the nucleus, a series of conserved
processing steps is required to produce mature mRNA competent
for translation in the cytoplasm.
• These nuclear processing steps include
1) 5′-end capping to generate a 7-methylguanosine
cap,
2) Splicing out of introns, and
3) 3′-end cleavage/polyadenylation to create a
mature, polyadenylated mRNA.
• Nuclear processing events are mediated by a myriad of RNA
binding proteins that associate with the nascent mRNA as soon
as the 5′-end emerges from RNA polymerase II.
• These RNA binding proteins contribute to packaging the RNA into
an mRNP complex competent for export to the cytoplasm.
 messenger ribonucleoprotein particles
(mRNPs)
• Control processing, export, localization, translation, and
degradation of mRNAs are necessary for regulation of
gene expression.
• These processes are controlled by
1. mRNA-specific regulatory proteins,
2. noncoding RNAs, and
3. core machineries common to most mRNAs
• mRNA concentrations have little correlation with
protein concentrations and that much of the
regulation of gene expression occurs at the level
of protein synthesis.
Consequences of Protein-mRNA
Interactions
Post-transcriptional steps in gene expression
• Export is achieved through the action of specific mRNA export
receptors that mediate interactions with the nuclear pore
complex.
• Remodeling of the mRNP complex at the cytoplasmic face of
the nuclear pore provides directionality to this movement
through the pore.
• Once in the cytoplasm, the mRNA can be
1) Translated to protein,
2) Stored in cytoplasmic bodies for future
translation,
3) Localized to specific regions of the cell, or
4) Targeted for decay.
• All these steps are fundamental to achieve gene expression and
support diverse cellular functions.
RNA binding proteins
• The vast majority of RNA binding proteins are
ubiquitously expressed.
• Only 6% of RNA binding proteins are expressed in a
tissue-specific manner.
• It mediates key steps in post-transcriptional regulation
of gene expression from mRNA processing to eventual
decay in the cytoplasm.
 Translation: DNA to mRNA to Protein
• The genes in DNA encode protein molecules, which are
the "workhorses" of the cell, carrying out all the
functions necessary for life.
• Expressing a gene means manufacturing its
corresponding protein, and this multilayered process has
two major steps.
1. In the first step, the information in DNA is transferred
to a messenger RNA (mRNA) molecule by way of a
process called transcription.
2. The resulting mRNA is a single-stranded copy of the
gene, which next must be translated into a protein
molecule.
A gene is expressed through the processes
of transcription and translation
Translation
• The second major step in gene expression, the mRNA
is "read" according to the genetic code, which relates
the DNA sequence to the amino acid sequence in
proteins.
• Each group of three bases in mRNA constitutes a
codon, and each codon specifies a particular amino
acid (hence, it is a triplet code).
• The mRNA sequence is thus used as a template to
assemble—in order—the chain of amino acids that
form a protein.
The amino acids specified by each mRNA codon

• The codons are

written 5' to 3', as
they appear in the
mRNA.
• AUG is an initiation
codon; UAA, UAG,
and UGA are
termination (stop)
codons.
• Multiple codons can
code for the same
amino acid.
 The ribosome
• Composed of two subunits: the large (50S) subunit and the small
(30S) subunit.
• Each subunit exists separately in the cytoplasm, but the two join
together on the mRNA molecule.
• The ribosomal subunits contain proteins and specialized RNA
molecules—specifically, ribosomal RNA (rRNA) and transfer RNA
(tRNA).
• The tRNA molecules are adaptor molecules—they have one
end that can read the triplet code in the mRNA through
complementary base-pairing, and another end that attaches to a
specific amino acid.
• Within the ribosome, the mRNA and aminoacyl-tRNA complexes
are held together closely, which facilitates base-pairing.
• The rRNA catalyzes the attachment of each new amino
acid to the growing chain.
 The Beginning of mRNA Is Not Translated
• An area near the 5' end of the molecule that is known as the
untranslated region (UTR) or leader sequence.
• This portion of mRNA is located between the first nucleotide that
is transcribed and the start codon (AUG) of the coding region, and
it does not affect the sequence of amino acids in a protein.
• The leader sequence is important because it contains a
ribosome-binding site.
• In human mRNA, the median length of the 5' UTR is about 170
nucleotides.
• If the leader is long, it may contain regulatory sequences,
including binding sites for proteins, that can affect the stability
of the mRNA or the efficiency of its translation.
 A DNA
Transcription
Unit.
 1. The
translation
initiation
complex
• The translation of mRNA begins
with the formation of a complex
on the mRNA.
• First, three initiation factor
proteins (known as IF1, IF2, and
IF3) bind to the small subunit of
the ribosome.
• This preinitiation complex and a
methionine-carrying tRNA then
bind to the mRNA, near the AUG
start codon, forming the initiation
complex.
 The large
ribosomal
subunit binds to
the small
ribosomal
subunit to
complete the
initiation
complex.
• The initiator tRNA molecule, carrying the
methionine amino acid that will serve as the
first amino acid of the polypeptide chain, is
bound to the P site on the ribosome.
• The A site is aligned with the next codon,
which will be bound by the anticodon of the
next incoming tRNA.
2. The Elongation Phase
• First, the ribosome moves along the mRNA in the 5'-to-
3'direction, which requires the elongation factor G, in a
process called translocation.
• The tRNA that corresponds to the second codon can
then bind to the A site, a step that requires
elongation factors as well as guanosine
triphosphate (GTP) as an energy source for the
process.
• Upon binding of the tRNA-amino acid complex in the A
site, GTP is cleaved to form guanosine diphosphate
(GDP), then released along with elongation factors for
the next round.
• Next, peptide bonds between the now-adjacent first and second
amino acids are formed through a peptidyl transferase
activity of rRNA.
• After the peptide bond is formed, the ribosome shifts, or
translocates, again, thus causing the tRNA to occupy the E site.
• The tRNA is then released to the cytoplasm to pick up another
amino acid. In addition, the A site is now empty and ready to
receive the tRNA for the next codon.
• This process is repeated until all the codons in the mRNA have
been read by tRNA molecules, and the amino acids attached to
the tRNAs have been linked together in the growing polypeptide
chain in the appropriate order. At this point, translation must be
terminated, and the nascent protein must be released from the
mRNA and ribosome.
3. Termination of Translation
• There are three termination codons that are employed at the end
of a protein-coding sequence in mRNA: UAA, UAG, and UGA.
• No tRNAs recognize these codons. Thus, release factors bind
and facilitates release of the mRNA from the ribosome and
subsequent dissociation of the ribosome.
• The translation process is very similar in prokaryotes and
eukaryotes. Although different elongation, initiation, and
termination factors are used, the genetic code is generally
identical.
• In bacteria, transcription and translation take place
simultaneously, and mRNAs are relatively short-lived.
• In eukaryotes, however, mRNAs have highly variable half-lives,
are subject to modifications, and must exit the nucleus to be
translated; these multiple steps offer additional opportunities to
regulate levels of protein production, and thereby fine-tune gene
expression.
 Control of Gene Expression
Programs
• The set of genes that are transcribed largely defines the cell.
• The gene expression program of a specific cell type includes RNA
species from genes that are active in most cells (housekeeping
genes) and genes that are active predominantly in one or a
limited number of cell types (cell-type-specific genes).
• The particular set of transcription factors that are expressed
in any one cell type controls the selective transcription of a subset
of genes by RNA polymerase II, thereby producing the gene
expression program of the cell.
• Only a small number of the transcription factors
that are expressed in cells are necessary to establish cell-type-
specific gene expression programs.
 Transcriptional Regulation
• DNA binding transcription factors (also known as trans-
factors) occupy specific DNA sequences at control
elements (cis-elements) and recruit and regulate the
transcription apparatus.
• Many different diseases and syndromes, including cancer,
autoimmunity, neurological disorders, diabetes,
cardiovascular disease, and obesity, can be caused by
mutations in regulatory sequences and in the
transcription factors, cofactors, chromatin
regulators, and noncoding RNAs that interact with
these regions.
• There are a remarkable variety and number of genes
that are transcribed into protein-coding and noncoding
RNA (ncRNA) species in mammalian cells.
• The human genome is thought to contain 20,000
protein-coding genes and at least as many ncRNA
genes.
• Many of the ncRNAs contribute to control of gene
expression through modulation of transcriptional or
posttranscriptional processes.
Transcriptional Control of Genes
Transcriptional regulation occurs at two
interconnected levels:
1) Transcription factors and the transcription
apparatus, and
2) Chromatin and its regulators
1. Transcription factors
• Transcription factors typically regulate gene expression
by binding enhancer elements and recruiting cofactors
and RNA polymerase II to target genes.
• Multiple transcription factors typically bind in a
cooperative fashion to individual enhancers and regulate
transcription from the core promoters of nearby or
distant genes through physical contacts that involve
looping of the DNA between enhancers and the core
promoters.
• Enhancers are occupied by nucleosomes with specific
modifications and are sensitive to DNase treatment.
• Approximately one million putative enhancers have
recently been identified in the human genome.
• More than half of the ∼1,200 genes encoding
transcription factors show some evidence of transcription
in ESCs.
• Only a few of these transcription factors are needed to
reprogram a broad range of cell types into induced pluripotent
stem cells (iPSCs).
• These ESC transcription factors, which include OCT4, SOX2, and
NANOG, are expressed at high levels, bind regulatory elements
and positively regulate their own gene expression through
interconnected autoregulatory loops.
• Activation of these endogenous interconnected autoregulatory
loops may be key to cellular reprogramming.
• Other cell types express cell-type-specific, or lineage-
specific, master transcription factors that are likely to
share these key properties of the ESC master transcription
factors.
A. Formation of a preinitiation complex
• Transcription factors bind to specific DNA elements (enhancers) and
to coactivators, which bind to RNA polymerase II, which in turn binds
to general transcription factors at the transcription start site.
• The DNA loop formed between the enhancer and the start site is
stabilized by cofactors such as the Mediator complex and
cohesin.
• The cohesin complex has circular dimensions capable of encircling
two nucleosome-bound molecules of DNA.
• The fundamental unit of chromatin, the nucleosome, is regulated
by protein complexes that can mobilize the nucleosome or
modify its histone components.
• Gene activation is accompanied by recruitment of ATP-dependent
chromatin remodeling complexes of the SWI/SNF family, which
mobilize nucleosomes to facilitate access of the transcription
apparatus and its regulators to DNA.
B. Initiation and pausing
• RNA polymerase II begins transcription from the
initiation site, but pause control factors cause it to
stall some tens of base pairs downstream.
• Once the recruited RNA polymerase II molecules
initiate transcription, they generally transcribe a
short distance, typically 20–50 bp, and then pause.
• This process is controlled by the pause control
factors DSIF and NELF, which are physically
associated with the paused RNA polymerase II
molecules.
C. Pause release and elongation

• Various transcription factors and cofactors recruit

elongation factors such as P-TEFb, which
phosphorylates the pause release factors and
polymerase, allowing elongation to proceed.
• D. Chromatin structure is regulated by ATP-dependent remodeling
complexes that can mobilize the nucleosome, allowing regulators
and the transcription apparatus increased access to DNA sequences.
• E. Transcriptional activity is influenced by proteins that modify and
bind the histone components of nucleosomes. Some proteins
add modifications (writers), some remove modifications (erasers),
and others bind via these modifications (readers). The modifications
include acetylation (Ac), methylation (Me), phosphorylation (P),
sumoylation (Su), and ubiquitination (Ub).
• F. Histone modifications occur in characteristic patterns
associated with different transcriptional activities. As an
example, the characteristic patterns observed at actively transcribed
genes are shown for histone H3 lysine 27 acetylation (H3K27Ac),
histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 79
dimethylation (H3K79me2), and histone H3 lysine 36 trimethylation
(H3K36me3).
• Repressed genes are embedded in chromatin with
modifications that are characteristic of specific repression
mechanisms.
• One type of repressed chromatin, which contains nucleosome
modifications generated by the Polycomb complex (e.g.,
histone H3K27me3), is found at genes that are silent but
poised for activation at some later stage of development and
differentiation.
• Another type of repressed chromatin is found in regions of the
genome that are fully silenced, such as that containing
retrotransposons and other repetitive elements.
• The mechanisms that silence this latter set of genes can involve
both nucleosome modification (e.g., histone
H3K9me3) and DNA methylation.
2. Chromatin Remodeling in
Eukaryotes
• The haploid human genome contains approximately 3 billion base
pairs of DNA packaged into 23 chromosomes.
• Histones are a family of small, positively charged proteins termed
H1, H2A, H2B, H3, and H4.
• DNA is negatively charged, due to the phosphate groups in
its phosphate-sugar backbone, so histones bind with DNA very
tightly.
• Nucleosomes are structured of two each of the histones H2A,
H2B, H3, and H4 come together to form a histone octamer, which
binds and wraps approximately 1.7 turns of DNA, or about 146
base pairs.
• The addition of one H1 protein wraps another 20 base pairs,
resulting in two full turns around the octamer, and forming a
structure called a chromatosome
Two major mechanisms by which chromatin
is made more accessible
1. Histones can be enzymatically modified by
the addition of acetyl, methyl, or phosphate
groups.
2. Histones can be displaced by chromatin
remodeling complexes, thereby exposing
underlying DNA sequences to polymerases and
other enzymes.
Dynamic properties of nucleosomes
 Histone
Modification and
the Histone Code
• Covalent bonding of
various functional
groups to the free
nitrogens in the R-
groups of lysines in
the N-terminal tail.
• acetylation,
phosphorylation,
methylation,
ubiquitination and
ADP-ribosylation that
take place on the
`tail' domains of
histones.
DNA methylation and gene silencing
• Heavily methylated regions of DNA with elevated
concentrations of these so-called CpG groups are often
found near transcription start sites.
• Proteins that bind to methylated DNA also form
complexes with proteins involved in deacetylation of
histones.
• Therefore, when the DNA is in a methylated state,
nearby histones are deacetylated, resulting in
compact, semi-permanently silent chromatin.
 Small Non-coding RNA and Gene
Expression
• siRNA and miRNA inhibit translation by two different mechanisms
while working in association with a protein, forming a
ribonucleoprotein complex called RNA-induced silencing complex
(RISC).
• The proteins in RISC unwind siRNA and remain bound to a single
antisense strand, which then binds to mRNA in a sequence-
specific manner, at which time a protein component of RISC
called Slicer cuts the mRNA in the middle of the binding region.
• The cut mRNA is recognized by the cell as being abnormal and is
subsequently destroyed. In the case of miRNA, a microRNA-
induced silencing complex (miRISC) associates with the mature
miRNA, and the complex binds to mRNA and physically blocks
translation.
• Many miRNAs form imperfectly complementary stem-loop
structures on the target sense strand of mRNA, as opposed to
siRNAs, which require near-perfect matches.
The mechanism of RNAi
Overview of the gene expression pathway
Contextual confounders of mRNA–protein correlations