1. Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan

2. Today’s lecture Gene expression
Constitutive, inducible, repressible genes
Specificity factors, activators, repressors
Negative and positive gene regulation
Lac operon
Helix-turn-helix motifs
Zinc-fingers
Leucine zippers

3. What is gene expression?
Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product.
A gene is expressed when its biological product is present and active.
Gene expression is regulated at multiple levels.

4. Regulation of gene expression
Promoter
Gene (red) with an intron (green)
Plasmid
single copy vs. multicopy plasmids
1. DNA replication
2. Transcription
Primary
transcript
mRNA degradation
3. Posttranscriptional
processing
Mature
mRNA
4. Translation
inactive
protein
Protein degradation
5. Posttranslational
processing
active
protein

5. Gene regulation (1) Condition 2 Condition 1 Chr. I Chr. II Chr. III
“turned
off”
on”
Condition 1
“turned
on”
“turned
off” 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Chr. I
Chr. II
Chr. III
constitutively
expressed gene
induced
gene
repressed
inducible/ repressible genes

6. Gene regulation (2) Condition 4 Condition 3 upregulated
gene expression
down regulated 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
constitutively
expressed gene

7. Definitions Constitutively expressed genes: Inducible genes:
Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells.
Inducible genes:
Genes that are transcribed and translated at higher levels in response to an inducing factor
Repressible genes:
Genes whose transcription and translation decreases in response to a repressing signal

8. Definitions Housekeeping genes:
genes for enzymes of central metabolic pathways (e.g. TCA cycle)
these genes are constitutively expressed
the level of gene expression may vary

9. Modulators of transcription
(1) specificity factors, (2) repressors, (3) activators
Specificity factors:
Alter the specificity of RNA polymerase
Examples: s-factors (s70, s32 ), TBPs
s70
s32
Standard
promoter
Housekeeping gene
Heat shock
promoter
Heat shock gene

10. Modulators of transcription
2. Repressors:
mediate negative gene regulation
may impede access of RNA polymerase to the promoter
actively block transcription
bind to specific “operator” sequences (repressor binding sites)
Repressor binding is modulated by specific effectors
Effector
(e.g. endproduct)
Repressor
Operator
Coding sequence
Promoter

11. Negative regulation (1)
Repressor
Effector
Example:
lac operon
RESULT:
Transcription occurs when the gene is derepressed
Source: Lehninger pg. 1076

12. Negative regulation (2)
Repressor
Effector (= co-repressor)
Example:
pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism
Source: Lehninger pg. 1076

13. Modulators of transcription
3. Activators:
mediate positive gene regulation
bind to specific regulatory DNA sequences (e.g. enhancers)
enhance the RNA polymerase -promoter interaction and actively stimulate transcription
common in eukaryotes
RNA pol.
Activator
promoter
Coding sequence

14. Positive regulation (1)
Activator
RNA polymerase
Source: Lehninger pg. 1076

15. Positive regulation (2)
Activator
Effector
RNA polymerase
Source: Lehninger pg. 1076

16. Operons
a promoter plus a set of adjacent genes whose gene products function together.
usually contain 2 –6 genes, (up to 20 genes)
these genes are transcribed as a polycistronic transcript.
relatively common in prokaryotes
rare in eukaryotes

17. The lactose (lac) operon
Contains several elements
lacZ gene = b-galactosidase
lacY gene = galactosidase permease
lacA gene = thiogalactoside transacetylase
lacI gene = lac repressor
Pi = promoter for the lacI gene
P = promoter for lac-operon
O1 = main operator
O2 and O3 = secondary operator sites (pseudo-operators) Pi P Z Y A I O3 O1 O2

18. into glucose and galactose.
The lac operon consists of three structural genes, and a promoter, a terminator,regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA.
lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose
into glucose and galactose.
lacY encodes β-galactoside permease (LacY), a membrane-bound transport protein that pumps lactose into the cell.
lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides.
Only lacZ and lacY appear to be necessary for lactose catabolism.
Theodor Hanekamp © 2003

19. First Level
The lacI gene coding for the repressor lies nearby the lac operon and is always expressed (constitutive).
Hinder production of β-galactosidase in the absence of lactose.
If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence called the lac operator.
The repressor binding to the operator interferes with binding of RNA Pol to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels.
When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose , which is a combination of glucose and galactose, binds to the repressor, causing a change in its shape.
Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.

20. Second Level
The second control mechanism is a response to glucose, which uses the Catabolite activator protein (CAP) to greatly increase production of β-galactosidase  in the absence of glucose. 
Cyclic adenosine monophosphate  (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose.
It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below),
which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase
enabling the cell to hydrolyse (digest) lactose and release galactose and glucose.
Theodor Hanekamp © 2003

21. Theodor Hanekamp © 2003

22. Regulation of the lac operonPi P Z Y A I Q3 Q1 Q2
LacZ
LacY
LacA
lacI repressor Pi P Z Y A I Q3 Q1 Q2
Inducer molecules:
Allolactose:
- natural inducer, degradable
IPTG (Isopropylthiogalactoside)
- synthetic inducer, not metabolized,

23. Selected DNA binding motifs
Helix-turn-helix
Homeodomain
Zinc Fingers
Cys4 zinc finger
Cys2 His2 zinc finger (e.g. TFIIIA)
Basic domains
Leucine zippers factors (bZIP)
Basic helix-loop-helix (bHLH)
Beta-scaffold factors with minor groove contacts
HMG (High mobility group) proteins

24. Helix-turn-helix motifs
Structure:
about 20 amino acids long
2 short alpha helicies ( 7 – 9 amino acids long)
DNA recognition helix (binds specific DNA sequence)
Recognition helix and 2nd helix form ~ 90° angle
very short turn ( NOT a beta-turn)
Often glycine at start of the turn (helix breaker)

25. How does the lac repressor bind DNA?
LacI repressor
(helix-turn-helix domain)
DNA recognition
helix
Second alpha helix
turn
DNA
Source: Lehninger pg. 1082

26. Zinc-Finger Motifs Several subtypes (Cys4, Cys2-His2 …)
Example: Cys2 His2 type
Zinc does not interact with DNA
Usually multiple zinc-fingers in a row
At least some also bind RNA
Consensus sequence:
[Y,F]-X-C-X2-4-C-XXX-F-XXXXX-L-XX-H-X3-5-H C H Zn C H Zn C H Zn

27. Basic domains Leucine zippers (bZip):
Basic region of the protein binds to DNA
Mainly act as dimers or other sometimes as other multimers
Special alpha-helices allow formation of coiled-coil structures.
Hydrophobic residues (Leu) align on one side of the helix
Example: Jun and Fos
abcdefg 7 7 7 7
Source: Lehninger pg. 1084

28. Leucine zippers
Leucines
DNA
Source: Lehninger pg. 1084

29. Transcription attenuation

30. Some Genes Are Regulated by Genetic Recombination
Example of Salmonella typhimurium

31. Regulation of Eukaryotic Gene Expression

32. Gene Regulation at DNA Level Chromatin Remodeling
1. Changes of DNA Topo structure Formation of ssDNA
DNase I hypersensitive site

33. 2. DNA Methylation
DNA Methylation

34.  CpG islands
----- are genomic regions that contain a high
frequency of CG dinucleotides.
----- CpG islands particularly occur at or near
the transcription start site of housekeeping genes.

35. Unmethylated CpG islandTF
RNA pol
Active transcription
Methylated CpG island TF
RNA pol
CH3
Repressed transcription

36. 3. Histone modification
 methylation
 acetylation TF

37. Functions of Histone methylation in transcription
Most well-studied histone modifications are involved in control of transcription.
Actively transcribed genes
Two histone modifications are particularly associated with active transcription:
Trimethylation of H3 lysine 4 (H3K4Me3) at the promotor of active genes
Trimethylation of H3 lysine 36 (H3K36Me3) in the body of active genes
Repressed genes
Three histone modifications are particularly associated with repressed genes:
Trimethylation of H3 lysine 27 (H3K27Me3)
Di and tri-methylation of H3 lysine 9 (H3K9Me2/3)
Trimethylation of H4 lysine 20 (H4K20Me3)

38. Functions of Histone methylation in transcription
Acetylated histones and nucleosomes represent a type of epigenetic tag within chromatin. Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.

39. 7.3 Transcriptional Regulation
1. Cis-acting element
(1) What is cis-acting element?
 Concept
Cis-acting elements - DNA sequences close to a gene that are required for gene expression

41. 2. What is trans-acting factor?
 Concept
trans-acting factors - usually they are proteins, that bind to the cis-acting elements to control gene expression.

42. These trans-acting factors can control gene expression in several ways:
 may be expressed in a specific tissue
 may be expressed at specific time in development
 may be required for protein modification
 may be activated by ligand binding

43. Domains of trans-acting factors
 DNA binding domain DBD
 transcription activating domain

44. Post-Transcriptional Regulation 1. Gene Regulation of mRNA Processing
exon shuffling
alternative gene splicing

45. 2. Gene Regulation of mRNA Editing
3. mRNA Longevity
mRNA Transport Control
RNA Interference (RNAi)
 miRNA
 siRNA

46. 7.5 Translational and Post-translational Regulation
1. Translation Control
Blocking mRNA Attachment to Ribosomes
2. Regulation of Protein Processing
Protein Modification

47. 3. Regulation of Protein Stability

48. Thank You Very Much