1. Ch 18.1-4, Campbell 9th edition
Gene regulation
Ch , Campbell 9th edition

2. Lac operon – inducible operon
18.1 Gene Regulation in Prokaryotes:
Lac operon – inducible operon
Normally, the repressor IS bound to the operator, so lac operon is OFF.
In the "induced" state, the lac repressor is NOT bound to the operator site.

3. Trp operon - repressible
Trp operon is normally ON
When tryptophan is present it binds to the repressor, which activates it. The activated repressor will bind to the operator, “repressing” the operon

4. Positive Gene Regulation
Positive activation of lac operon
Bacteria uses carbohydrates for energy – glucose vs. lactose
How is this signaled to the bacteria?

5. Positive activation is through the CAP protein, which is activated by cAMP.
cAMP accumulates when glucose is scarce.

6. 18.2 Gene Regulation in Eukaryotes
Gene is expressed when it makes a protein
Expression regulated at various levels:
Chromatin structure
Transcription factors*
Alternative splicing
Non-coding RNAs that degrade other mRNAs

7. Chromatin packing

8. Chromatin structure
Histone acetylation- acetyl groups are added to histones
loosens chromatin structure – promotes transcription
Deacetylation – acetyl groups removed, reduces transcription
Methylation – methyl groups added to certain bases in DNA
Reduces transcription in some species
In genomic imprinting, regulates expression of maternal or paternal alleles of certain genes

10. Regulation of Transcription: Typical Eukaryotic Gene Organization
Control elements, segments of noncoding DNA, are associated with eukaryotic genes. Control elements act as binding sites for transcription factors that help regulate transcription
Control elements and the transcription factors that bind them allow for precise control of gene regulation

11. Eukaryotic gene & transcript
Enhancer (distal control elements)
DNA
Upstream
Promoter
Proximal control elements
Transcription start site
Exon
Intron
Poly-A signal sequence
Transcription termination region
Downstream
Poly-A signal
Transcription
Cleaved 3 end of primary transcript 5
Primary RNA transcript (pre-mRNA)
Intron RNA
RNA processing
mRNA
Coding segment
5 Cap
5 UTR
Start codon
Stop codon
3 UTR 3
Poly-A tail P G
AAA  AAA

12. Transcription factors – general ones are required for the RNA polymerase binding. They bind first to the DNA, and then recruit the RNA polymerase. Specific transcription factors bind with control elements for regulation
Enhancers – groups of control elements upstream of a gene, have binding sites for specific transcription factors

13. Activators - a protein that binds to an enhancer and stimulates transcription of a gene
have two domains, one that binds DNA and a second that activates transcription
Repressors - transcription factors that inhibit expression of a particular gene by a variety of methods

14. MyoD – a specific transcription factor that acts as an activator
MyoD is a master regulatory gene
DNA
Activation domain
DNA-binding domain

15. Gene Switches

16. Activators
DNA
Enhancer
Distal control element
Promoter
Gene
TATA box
General transcription factors
DNA- bending protein
Group of mediator proteins
RNA polymerase II
RNA synthesis
Transcription initiation complex

17. Model for Transcription Initiation
1. Transcriptional activators bind to DNA & recruit chromatin remodeling complexes and histone acetyltransferases
2. These open up the chromatin to expose promoter & regulatory sequences
3. Transcriptional factors bind to enhancers
4. DNA bending protein protein brings activators, mediator proteins, and general transcirption factors together to form transcription initiation complex on promoter

18. Regulation of Eukaryotic DNA Transcription

19. Coordinately controlled genes in eukaryotes
Genes coding for enzymes of a metabolic pathway are often scattered over different chromosomes
Coordinate gene expression depends on simultaneous expression of the genes
Chemical signalling is often used for coordinate gene expression – i.e. hormones

20. Alternate Gene Splicing
Post transcriptional regulation through RNA processing
Different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

21. 1 2 3 4 5 Exons DNA Troponin T gene Primary RNA transcript
RNA splicing or
mRNA 1 2 3 4 5

22. mRNA degradation
Lifespan of mRNA in cytoplasm affects protein synthesis
mRNA in eukaryotes lasts longer than prokaryotic mRNA

23. Translation
Initiation of translation can be blocked by proteins that bind to parts of mRNA

24. Protein processing & degradation
Post-translational protein processing includes cleavage, and addition of functional groups
Proteasomes are giant protein complexes that bind protein molecules and degrade them

26. 18.3 Non coding mRNAs
A large part of the genome is made up of DNA that is transcribed into non-coding mRNAs (ncRNA)
These can affect translation and chromatin expression

27. RNA
Important in many cellular machines:
Ribosome rRNA
Spliceosome snRNA
Telomerase telomerase RNA

28. Interference with Translation
MicroRNAs (miRNA) are single-stranded RNA molecules that can bind to mRNA
Small Interfering RNA (siRNA) act similarly to miRNAs, but have a longer, double stranded precursor
They can degrade mRNA or block its translation
This is called RNA interference - RNAi

29. (a) Primary miRNA transcript Hairpin miRNA Hydrogen bond
Dicer
miRNA- protein complex
mRNA degraded
Translation blocked
(b) Generation and function of miRNAs 5 3

30. microRNAs A novel class of ncRNA gene Products are ~22 nt RNAs
Precursors are nt hairpins
Gene regulation by pairing to mRNA
Unknown before 2001
Forms RISC – RNA inducing silencing complex

31. Small Interfering RNAs - siRNA
RNA interference (RNAi) – when double stranded RNA injected into the cell, it turned off expression of gene with same sequence as the RNA
siRNAs are the cause of this RNAi
Similar to miRNA, but formation is different
Many siRNAs are formed from a longer, double stranded RNA molecule
Some siRNAs can bind back to chromatin and cause changes in the chromatin

32. siRNA

33. Chromatin & ncRNA
In some yeasts, siRNAs can play role in heterochromatin forming, and block parts of chromosome
Small ncRNAs can induce heterochromatin, which blocks parts of chromosome, blocking transposons

34. RNAi (~5 min)]

35. 18.4 Differential gene expression leads to different cell types in multicellular organism
One fertilized egg can give rise to many different cell types
Differential gene expression results from genes being regulated differently in each cell type
Materials in the egg can set up gene regulation that is carried out as cells divide

36. Cell development
Zygote cell – totipotent – has potential to develop into a complete organism
Cell determination – cell has committed to a final fate, it is unable to change at this point
Cell differentiation – cell produces tissue-specific proteins, cell has clear cut identity
Morphogenesis – organization of cells into tissues & organs

37. Cytoplasmic Determinants
Based on uneven distribution of cytoplasmic determinants in egg
The cytoplasm has RNA & proteins that were encoded by the mother’s DNA
When cell divides, the two cells have different amounts of the determinants, which can determine the cell’s fate

38. (a) Cytoplasmic determinants in the egg
Unfertilized egg
Sperm
Fertilization
Zygote (fertilized egg)
Mitotic cell division
Two-celled embryo
Nucleus
Molecules of two different cytoplasmic determinants

39. Induction signals and cell differentiation
Environment around the cell, especially signals from nearby embryonic cells influence development of cells
The changes in gene expression lead into observable cellular changes
In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells
Interactions between cells induce differentiation of specialized cell types

40. Signal Induction - types
Signals from one group of cells influence another group of cells
Diffusion: signal diffuses from distance to receptor– i.e. hormone, or other signal molecule
- receptor can transmit signal through second messengers in signal transduction pathway

41. Signal Induction - types
Direct contact – neighboring cells
Gap junction – cytoplasm of 2 cells is connected

42. Muscle cell determination

43. Pattern formation
What controls the body plan of an organism? How do organs get in the right place?
2 general models:
Morphogen gradient
Sequential induction

44. Sequential induction
Differentiation due to production & release of a series of chemical signals

45. Morphogen gradient
A diffusible chemical signal, or morphogen, is produced.
The concentration is higher closer to the source, and lower farther away from the source.
The fate of the cell depends on its exposure to the different threshold levels.

46. Drosophila- model organism
Lewis studied development by looking at mutants with bizarre developmental defects, and through this discovered homeotic genes
Homeotic genes specify the identity of body segments
Mutations in these genes lead to structures in the wrong place

47. In a fruit fly, for example, Hox genes lay out the various main body segments—the head, thorax, and abdomen. Here we see a representation of a fruit fly embryo viewed from the side, with its anterior end to the left and with various Hox genes shown in different colors. Each Hox gene, such as the blue Ultrabithorax or Ubx gene, is expressed in different areas, or domains, along the anterior-to-posterior axis.

48. Drosophila development
Cytoplasmic determinants in egg establish axes of drosophila body
Bicoid mRNA from mother is translated into the Bicoid protein in the Drosophila zygote
Bicoid is transcription factor that turns on genes in different levels

49. a. Bicoid concentration & 4 genes affected
b. concentration gradient of Bicoid in zygote– more at right
c. concentration gradient in embryo after several divisions
d. hunchback protein – green, kruppel protein - orange

50. Eric Wieschaus – Bicoid gradient (3:28)
Bicoid animation (2:15)

51. Bonnie Bassler – Quorum sensing & gene expression

52. miRNA slides from: