Mechanism of gene regulation

1. K. Narayanapura, Kothanur (PO), Bengaluru 560077
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e-mail:info@kristujayanti.com, www.kristujayanti.edu.in
Regulation of gene expression in
Eukaryotes
Dr. Manikandan Kathirvel
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Bengaluru

2. Regulation of Gene Expression in
Eukaryotes
Gene expression is the combined process of :
1. the transcription of a gene into mRNA,
2. the processing of that mRNA, and
3. its translation into protein (for protein-encoding genes).
Levels of regulation of gene expression

3. Purpose of regulation of gene expression:
Regulated expression of genes is required for:
1)Adaptation- Cells of multicellular organisms respond to varying
conditions. Such cells exposed to hormones and growth factors
change substantially in – shape, growth rate and other characteristics
2) Tissue specific differentiation and development
•The genetic information present in each somatic cell of a organism is
practically identical.
•Cells from muscle and nerve tissue show strikingly different morphologies and
other properties, yet they contain exactly the same DNA.
•These diverse properties are the result of differences in gene
expression.
•Expression of the genetic information is regulated during
developmental stage of an organism and during the differentiation of
tissues and biologic processes in the multicellular organism.
•Transcription control can result in tissue-specific gene expression.

4. Differences between prokaryotic and eukaryotic gene expression:
1) Absence of operons: Prokaryote gene expression typically is
regulated by an operon, the collection of controlling sites adjacent
to polycistronic protein-coding sequences.
• Eukaryotic genes also are regulated in units of protein-coding
sequences and adjacent controlling sites, but operons are not
known to occur.
2.) Complexity: Eukaryotic gene regulation is more complex because
eukaryotes possess a nucleus.
3) Different cell types: Different cell types are present in most
eukaryotes. Liver and pancreatic cells, for example, differ dramatically
in the genes that are highly expressed. Different mechanisms are
involved in the regulation of such genes.
4.) Uncoupled transcription and translation processes: transcription and
translation are not coupled).
i. In prokaryotes, transcription and translation are coupled processes, the primary transcript is
immediately translated.
ii. The transcription and translation are uncoupled in eukaryotes, eliminating some potential
generegulatory mechanisms.
iii. The primary transcript in eukaryotes undergoes modifications to become a mature functional m RNA.

5. 5) Chromatin structure (in eukaryotes)
- The DNA in eukaryotic cells is extensively folded and packed into the
protein-DNA complex called chromatin.
Histones are an important part of this complex since they both form
the structures known as nucleosomes and also contribute significantly
into gene regulatory mechanisms.
- Heterochromatin (tanscriptionally inactive) and Euchromatin
(transcriptionally active)
Regulation of gene expression in eukaryotes:
Two “categories” of eukaryotic gene regulation exist:
 Levels of control of gene expression
Short term control:
Genes are quickly turned on or off in response to the
environment and demands of the cell.
(to meet the daily needs of the organism)
Long term control
(genes for development/differentiation)

6. Mechanism of regulation of gene expression- An
overview
•Gene activity is controlled first and foremost at the level of
transcription.
•Much of this control is achieved through the interplay
between proteins that bind to specific DNA sequences and
their DNA binding sites.
•This can have a positive or negative effect on transcription.
•Transcription control can result in tissue-specific gene
expression.
•In addition to transcription level controls, gene
expression can also be modulated by translational
level.
Eukaryotic gene expression is controlled by:
•Cis-Trans acting elements- Transcriptional activation/
repression
•Chromatin modifications
•Gene rearrangement,
•Gene amplification,
•Posttranscriptional modifications, and
•RNA stabilization
•Regulation by noncoding RNA
•Post translational modifications
•Protein Degradation

7. 1. Cis-Trans acting elements: Transcriptional activation/Repression:
Transcriptional control of gene regulation is controlled by:
a) Promoters
b) Enhancers
c) Silencers
d) Transcriptional factors
e) Activators
f) repressors

8. 1) Cis and Trans acting elements:
Promoters
1. Transcriptional activation:

9. Promoters

10. Promoters
• Occur upstream of the transcription start site.
• Some determine where transcription begins (e.g., TATA),
whereas others determine if transcription begins.
• Promoters are activated by specialized transcription factor (TF)
proteins (specific TFs bind specific promoters).
• Transcription of rRNA – Transcriptional factor- I
• Transcription of mRNA- Transcriptional factor- II
• Transcription of tRNA- Transcriptional factor -III
• One or many promoters (each with specific TF proteins) may
occur for any given gene.
• Promoters may be positively or negatively regulated.

11. Enhancers and Repressors
i. Enhancer elements are regulatory DNA sequences, although they have no promoter activity of
their own but they greatly increase the activities of many promoters in eukaryotes.
ii. Enhancers function by serving as binding sites for specific regulatory proteins such as
transcriptional factors., when bound by transcription factors, enhance the transcription of an
associated gene.
iii. An enhancer is effective only in the specific cell types in which appropriate regulatory proteins
are expressed.
iv. Enhancer elements can exert their positive influence on transcription even when separated by
thousands of base pairs from a promoter;
v. they work when oriented in either direction; and they can work upstream (5') or downstream (3')
from the promoter.
vi. Enhancers are promiscuous; they can stimulate any promoter in the vicinity and may act on more
than one promoter.
• Occur upstream or downstream of
the transcription start site or within
the coding sequence
• Regulatory proteins bind specific
enhancer sequences; binding is
determined by the DNA sequence.
• Interactions of regulatory proteins
determine if transcription is activated
or repressed (positively or negatively
regulated).

12. Enhancers and Transcription
In some eukaryotic genes, there are regions
that help increase or enhance transcription.
These regions, called enhancers, are not
necessarily close to the genes they enhance.
They can be located upstream of a gene, within
the coding region of the gene, downstream of a
gene, or may be thousands of nucleotides
away.
Enhancer regions are binding sequences, or
sites, for transcription factors.
When a DNA-bending protein binds to an
enhancer, the shape of the DNA changes. This
shape change allows the interaction between
the activators bound to the enhancers and the
transcription factors bound to the promoter
region and the RNA polymerase to occur.
Therefore, a nucleotide sequence thousands of
nucleotides away can fold over and interact
with a specific promoter.
An enhancer is a DNA sequence that promotes
transcription. Each enhancer is made up of
short DNA sequences called distal control
elements. Activators bound to the distal
control elements interact with mediator
proteins and transcription factors.

14. Silencers:
•The elements that decrease or repress the expression of specific genes have
also been identified called Silencers.
•Silencers are control regions of DNA that, like enhancers, may be located
thousands of base pairs away from the gene they control.
•However, when transcription factors bind to them, expression of the gene they
control is repressed.

15. Trans acting elements

19. MYC gene encodes a multifunctional,
nuclear phosphoprotein that controls a
variety of cellular functions, including
cell cycle, cell growth, apoptosis,
cellular metabolism and biosynthesis,
adhesion, and mitochondrial
biogenesis.

20. Regulation of gene expression in eukaryotes:
Two “categories” of eukaryotic gene regulation exist:
 Levels of control of gene expression
Short term control:
Genes are quickly turned on or off in response to the
environment and demands of the cell.
(to meet the daily needs of the organism)
Long term control
(genes for development/differentiation)

21. Short-term - transcriptional control of
galactose-utilizing genes in yeast:
• 3 genes (GAL1, GAL7, & GAL 10) code
enzymes that function in the galactose
metabolic pathway.
• GAL1 galactokinase
• GAL7 galactose transferase
• GAL10 galactose epimerase
• Pathway produces d-glucose 6-phosphate,
which enters the glycolytic pathway and is
metabolized by genes that are
continuously transcribed.
• In absence of galactose, GAL genes are not
transcribed.
• GAL genes rapidly induced by galactose
and absence of glucose.
• Analagous to E. coli lac operon repression
by glucose.

23. Galactose metabolizing pathway of yeast.

24. Short-term - transcriptional control of galactose-utilizing genes in yeast:
• GAL genes are near each other but do not constitute an operon.
• Additional unlinked gene, GAL4, and GAL80 codes a repressor
protein that binds a promoter element called an upstream activator
sequence (UASG).
• UASG is located between GAL1 and GAL10.
• Transcription occurs in both directions from UASG.
Conditions:
• When galactose is absent, the GAL4 product (GAL4p) and another
product (GAL80p) bind the UASG sequence; transcription does not
occur.
• When galactose is added, a galactose metabolite binds GAL80p and
GAL4p amino acids are phosphorylated.
• Galactose acts as an inducer by causing a conformation change in
GAL4p/GAL80p.

25. Activation model of GAL genes in yeast.
Conditions:
• When galactose is absent, the GAL4
product (GAL4p) and another product
(GAL80p) bind the UASG sequence;
transcription does not occur.
Conditions:
• When galactose is added, a galactose
metabolite binds GAL80p and GAL4p amino
acids are phosphorylated.
• Galactose acts as an inducer by causing a
conformation change in GAL4p/GAL80p.

26. Conditions:
• When galactose is absent, the GAL4 product (GAL4p) and another product
(GAL80p) bind the UASG sequence; transcription does not occur.

27. Conditions:
• When galactose is added, a galactose metabolite binds GAL80p and
GAL4p amino acids are phosphorylated.
• Galactose acts as an inducer by causing a conformation change in
GAL4p/GAL80p.

28. 2) Chromatin Remodeling
• Chromatin structure provides an important level of control of gene
transcription.
• The development of specialized organs, tissues, and cells and their function in
the intact organism depend upon the differential expression of genes.
• Some of this differential expression is achieved by having different regions of
chromatin available for transcription in cells from various tissues.
Large regions of chromatin are
transcriptionally inactive in some cells,
while they are either active or
potentially active in other specialized
cells.
For example, the DNA containing the -
globin gene cluster is in "active"
chromatin in the reticulocytes but in
"inactive" chromatini n muscle cells.

29. Formation and disruption of
nucleosome structure:
• The presence of nucleosomes
and of complexes of histones and
DNA provide a barrier against the
ready association of transcription
factors with specific DNA regions.

30. The disruption of nucleosome structure
is therefore an important part of
eukaryotic gene regulation and the
processes involved are as follows:
i) Histone acetylation and deacetylation
Acetylation is known to occur on lysine
residues in the amino terminal tails of
histone molecules.
This modification reduces the positive
charge of these tails and decreases the
binding affinity of histone for the
negatively charged DNA.
Accordingly, the acetylation of histones
could result in disruption of
nucleosomal structure and allow
readier access of transcription factors
to cognate regulatory DNA elements.

31. ii) Modification of DNA
Methylation of deoxycytidine residues in DNA may effect gross changes
in chromatin so as to preclude its active transcription.
Example: Acute demethylation of deoxycytidine residues in a specific region of
the tyrosine aminotransferase gene—in response to glucocorticoid hormones—has
been associated with an increased rate of transcription of the gene.

33. iii) DNA binding proteins
•The binding of specific transcription factors to certain DNA elements may result
in disruption of nucleosomal structure.
•Many eukaryotic genes have multiple protein-binding DNA elements.
•The serial binding of transcription factors to these elements may either directly
disrupt the structure of the nucleosome or prevent its re-formation.
•These reactions result in chromatin-level structural changes that in the
end increase DNA accessibility to other factors and the transcription
machinery.

34. 3) Gene Amplification
The gene product can be increased by increasing the number of genes available
for transcription of specific molecules
During early development of metazoans, there is an abrupt increase in the need
for ribosomal RNA and messenger RNA molecules (hundreds of copies of
ribosomal RNA genes and tRNA genes) for proteins that make up such organs as
the eggshell.
Such requirements are fulfilled by amplification of these specific genes.
Subsequently, these amplified genes, presumably generated by a process of
repeated initiations during DNA synthesis, provide multiple sites for gene
transcription.
Gene amplification has been demonstrated in patients receiving methotrexate for
cancer.
The malignant cells can develop drug resistance by increasing the number of
genes for dihydrofolate reductase, the target of Methotrexate.

35. 4.) mRNA stability
Although most mRNAs in mammalian cells are very stable (half-lives
measured in hours), some turn over very rapidly (half-lives of 10–30
minutes).
mRNA stability is subject to regulation.
This has important implications since there is usually a direct
relationship between mRNA amount and the translation of that mRNA
into its cognate protein.
Changes in the stability of a specific mRNA can therefore have major
effects on biologic processes.
The stability of the mRNA can be influenced by hormones and certain
other effectors.
The ends of mRNA molecules are involved in mRNA stability.
The 5' cap structure in eukaryotic mRNA prevents attack by 5'
exonucleases, and the poly(A) tail prohibits the action of 3'
exonucleases.
Poly A tailing- prevent the mRNA from degradation and gives stability
and helps the mRNA to transport from nucleus to cytoplasm
RNA splicing

36. 5. Regulation of gene expression by Noncoding RNA
A. Different regions of
mRNA that can be
targeted by an ncRNA
B. Mechanisms of
translation regulation
mediated by ncRNAs
C. RNase E and RNase III
dependent mRNA
degradation mediated by
ncRNAs

37. 6. Alternative splicing
7. Gene rearrangement
8. Post translational modifications
9. Protein Degradation by proteasome complex (Ubiquitination)

38. Summary and contrasts:
Prokaryotes control expression by:
Transcription
Eukaryotes control expression by:
Transcription
RNA processing
mRNA transport
mRNA translation
mRNA degradation
Protein degradation
Fig. 18.1

39. Thank you