1. The University of Texas-Houston,USA
MAHATMA GANDHI MISSION'S
Department of Infectious Diseases
In technical collaboration with
The University of Texas-Houston,USA
Regulation of Gene
Expression
Mr. Malcolm Nobre, DGM Texas Biotech
MGM University of Health Sciences
Department of Biotechnology

2. Objectives To know and explain:
Regulation of Bacterial Gene Expression
Constitutive ( house keeping) vs. Controllable genes
OPERON structure and its role in gene regulation
Regulation of Eukaryotic Gene Expression at different levels:
DNA methylation
Histon modifications(Chromatin Remodeling)
Increasing the number of gene copies (gene amplification)
Changing the rate of initiation of transcription
Alternate splicing
mRNA stability
Changing the rate of initiation of translation
Using of Untranslating Region (UTR) protein stability
Hormonal regulation
Cross talk between different regulatory pathways
11-Regulation by protein stability

3. Classification of gene with respect to their Expression
Constitutive ( house keeping) genes:
1- Are expressed at a fixed rate, irrespective to the cell condition.
2- Their structure is simpler
Controllable genes:
1- Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
2- Their structure is relatively complicated with some response elements

4. Gene Regulation
in Prokaryotes

5. Different ways for regulation of gene expression in bacteria
1- Promoter recognition:
2-Transcription elongation( Attenuation)

6. OPERON in gene regulation of prokaryotes
Definition: a few genes that are controlled collectively by one promoter
Its structure: Each Operon is consisted of few structural genes( cistrons) and some cis-acting element such as promoter (P) and operator (O).
Its regulation: There are one or more regulatory gene outside of the Operon that produce trans-acting factors such as repressor or activators.
Classification:
1- Catabolic (inducible) such as Lac OPERON Anabolic (repressible) such as ara OPERON
3- Other types

7. General structure of an OPERON

8. The activity of an Operon in the presence or the absence of repressor
No repressor
With repressor
Figure 8.13

9. Lac OPERON an inducible Operon
In the absence of lac
In the presence of lac

10. CRP or CAP is positive regulator of Lac and some other catabolic Operons
In the presence of lac + glucose
CRP= Catabolic gene regulatory Protein
CRP= cAMP receptor Protein
CAP= Catabolic gene Activating Protein

11. Trp OPERON a repressible example
In the absence of Trp
In the presence of Trp

12. Starved: antitermination Nonstarved: termination
Attenuation by different secondary RNA structure
Starved: antitermination
Nonstarved: termination

13. The attenuators of some operons36

14. Gene Regulation
in Eukaryotes

15. Eukaryotic gene regulation occurs at several levels

16. 1- Control at DNA level by DNA methylation
Heterochromatin is the most tightly packaged form of DNA. transcriptionally silent, different from cell to cell
Methylation is related to the Heterochromatin formation
Small percentages of newly synthesized DNAs (~3% in mammals) are chemically modified by methylation.
Methylation occurs most often in symmetrical CG sequences.
Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes.
Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation.

17. 2- Control at DNA level by Histon modifications(Chromatin Remodeling)
Acetylation by HATs and coactivators leads to euchromatin formation
Methylation by HDACs and corepressors leads to heterochromatin formation

18. 3-Control at DNA level by gene amplification
Repeated rounds of DNA replication yield multiple
copies of a particular chromosomal region.

19. 4- Control at transcription initiation
By using different sequences (promoter, enhancer or silencer sequences) and factors, the rate of transcription of a gene is controlled
gene X
promoter
gene control region for gene X

20. 5- Control at mRNA splicing (alternate splicing)
(four exons) 1 2 3 4
1, 2 & 4
1, 2 & 3
cell 2
cell 1
Calcitonin gene-related peptide 61
32 amino acids
Reduces bone resorption
37 amino acids
Vasodilator

21. 5- Alternative splicing: A Role in Sexual Behavior in Drosophila
a. In Drosophila courtship, the male behaviors include: Following, Singing & …
b. Regulatory genes (fruitless= fru) in the sex determination pathways control these behaviors.
c. Physiologically, the CNS (central nervous system) is responsible for key steps in male courtship behavior.) (fruitless)
The sex-specific fru mRNAs are synthesized in only a few neurons in the CNS (500/100,000). The proteins encoded by these mRNAs regulate transcription of a set of specific genes, showing that fru is a regulatory gene. Its expression seems to be confined to neurons involved in male courtship

22. 6- Control at mRNA stability
The stem loop at 3’end is an’ iron response element’.
The stem loop is stabilised by a 90 kDa protein in the absence of iron and protects the mRNA from degradation.
90 kDa iron sensing protein (aconitase)
No iron :
mRNA is translated into protein
Transferrin receptor
mRNA
AUG
UAA
+ iron
For the iron (Fe 2+) transport protein transferrin receptor.
A stem loop structure in the mRNA acts as an iron response element and binds a 90 kDa protein in the absence of iron. The RNA and iron binding regions of the protein overlap so in the presence of iron the 90 kDa binding protein can no longer bind to the mRNA iron response element and the stem loop no longer occurs. Since the stem loop is at the 3’ end of the mRNA , the loop is stabilising of the mRNA , protecting it from degradation. In the presence of iron, the loop disappears and the mRNA is degraded by 3’ exonucleases.s Fe
+ iron
stimulates
Transferrin receptor mRNA
Degraded by 3’ nuclease
In the presence of iron, transferrin receptor protein synthesis is reduced.

23. 6- Control at mRNA stability
A stem loop is stabilised by the 90 kDa protein in the absence of iron.
This time, the stem loop is at the 5’ end of the mRNA.
No iron
Ferritin mRNA
AUG
The presence of the stem loop prevents translation of this mRNA by blocking the progress of the ribosomes along the mRNA.
The binding of iron to the 90 kDa protein has opposite effects for ferritin. In this case, the stem loop is at the 5’ end. It inhibits translation by preventing ribosomes getting onto the mRNA and thus its disappearance stimulates transcription. Its removal leads to degradation of the mRNA and thus reduces translation.
+ iron Fe
+ iron
stimulates
AUG
UAA
In the presence of iron, the hairpin is lost, the ribosomes can translate the mRNA and ferritin protein synthesis is increased.

24. 6- Control at mRNA stability
Some hormones which enhance the production of proteins also increase the half life of the protein’s mRNA.
Estrogen : ovalbumin t1/2 from 2- 5hr to >24hr
Prolactin : casein t1/2 from 5 hr to 92hr
mRNA stability. When milk protein synthesis is stimulated in the mammary epithelium at child birth, the rapid increase in casein level arising from the pituitary hormone prolactin results from increased transcription of the casein gene but also from stabilisation of its mRNA. In fact, the stabilisation of mRNA is an essential component of the rapid build up of casein protein and this sort of regulation is evident in many situations in which the production of a particular protein needs to be increased to a high level.
The mechanism is not well understood. The poly A tail protects the mRA from 3’ degradation. Histone mRNA (histones are produced during the DNA synthetic phase of the cell cycle) do not have a poly(A) tail and are unstable. The sequence of bases in the 3’ untranslated region, especially runs of As and Us can affect the stability of individual mRNAs. However, the enzzymes that break down the RNA are not well characterised.

25. 7- Control at initiation of translation
5’ UTR
3’ UTR
AUG
UAA
Specific sequences make specific secondary structures
Specific protein factors bind to these secondary structures

26. 8-Regulation by protein stability
Ubiquitin-dependent proteolysis. Cyclins control of cell cycle.
Protein molecule is tagged for degradation by attachment of a 20 kDa protein, ubiquitin
ATP CO NH
NH2 +
COOH CO NH
26S proteasome
Doomed protein molecule
ubiquitin protein ligase
Protein stability. Ubiquitin system.
20 kda protein ubiquitin is activated by ATP
It is linked by its C terminus to amino group on a lysine side chain in target protein.
Enzyme is ubiquitin protein ligase.
Up to 50 molecules of ubiquitin / target protein molecule.
Ubiquitinylated protein molecule then degraded by proteosome. A large multiprotien complex (2000 kDa)
The level of cell cycle regulatory proteins called cyclins are produced at the G1 to S phase boundary of the cell cycle. The proteins stimulate kinases which trigger DNA synthesis. Once this triggering has occurred, there is no further need for the cyclins and they are degraded by the ubiquitin system. This system allows for rapid removal of proteins. It is selective. Another method for degrading proteins, the lysozome is non-selective.
It was originally thought that breakdown of proteins occurred in the lysozomes. However, reticulocytes which do not have lysozomes still break down abnormal proteins. The system involves a 76 amino acid residue protein called ubiquitin, because it is widespread in eukaryotic spp. It is also one of the most conserved proteins known, differing in only 3 AA between human and fruit fly.
Attachment is to the C terminus of ubiquitin and this is transferred to the amino group of a lysine side chain of the protein. Many ubiquitins per target protein molecule.
Proteasome is a 20 s/u multi protein complexcontaining at least 5 different proteolytic activities in the shape of a bi-capped hollow barrel. UBIQUITIN IS NOT DEGRADED.
The stability of a protein depends upon its N-terminal amino acid (the N-end rule).
N-terminal : For example arginine , lysine : protein t1/2 = 3 min
N-terminal : For example methionine, alanine, : t1/2 >20 hrs.

27. Regulation by water soluble Hormones
Polypeptide hormones bind at the cell surface and activate
transmembrane enzymes to produce second messengers
(such as cAMP) that activate gene transcription.

28. Regulation by water soluble Hormones

29. Regulation by lipid soluble Hormones
Steroid hormones pass through the cell membrane
and bind cytoplasmic receptors, which together
bind directly to DNA and regulate gene expression.