1. Regulation of Gene Expression
Chapter 18

2. Overview of Gene Expression
Regulation of Gene Expression
Overview of Gene Expression
The control of gene expression is vital to the proper and efficient functioning of an organism.
Cells control metabolism by either regulating enzyme activity –or- regulating the expression of genes coding for enzymes.

3. Figure 18.2 Figure 18.2 – Regulation of a Metabolic Pathway
In this example of the pathway of tryptophan synthesis, an abundance of tryptophan can either
Inhibit the activity of the first enzyme in the pathway (feedback inhibition)
Repress expression of the genes encoding all subunits of the enzymes in the pathway

4. Control of Gene Expression in Bacteria
Prokaryotic Gene Regulation
Control of Gene Expression in Bacteria
Bacteria often respond to environmental change by regulating transcription.
In bacteria, genes are often clustered into operons, with one promoter serving several adjacent genes.
An operator site on the DNA switches the operon on or off, resulting in coordinate regulation of the genes.

5. Operons: The Basic Concept
Prokaryotic Gene Regulation
Operons: The Basic Concept
An operon is essentially a set of genes and the switches that control the expression of those genes.
An operon consists of:
operator
promotor
and genes that they control
All together, the operator, the promoter, and the genes they control – the entire stretch of DNA required for enzyme production for the pathway – is called an operon.

6. Prokaryotic Gene Regulation
The Operon Model
Promoter Region: a base sequence that signals the start site for gene transcription; this is where RNA polymerase binds to begin the process.
Operator: a short sequence near the promoter that assists in transcription by interacting with regulatory proteins (transcription factors).
Repressor: protein that prevents the binding of RNA polymerase to the promoter site.
Enhancer: DNA region, also known as “regulator”, that is located thousands of bases away from the promoter; it influences transcription by interacting with specific transcription factors.
Inducer: a molecule that binds to and inactivates a repressor (i.e. lactose for the lac operon).
Operon: a promoter/operator pair that services multiple genes.

7. Repressible & Inducible Operons
Prokaryotic Gene Regulation
Repressible & Inducible Operons
There are basically two types of operons found in prokaryotes: repressible operons and inducible operons.
Both the repressible and inducible operon are types of NEGATIVE gene regulation because both are turned OFF by the active form of the repressor protein.
In either type of operon, binding of a specific repressor protein to the operator shuts off transcription.
Trp operon – repressible operon is always in the on position until it is not needed and becomes repressed or switched off.
Lac operon – inducible operon is always off until it is induced to turn on.

8. Figure 18.3a – The trp Operon
Tryptophan is an amino acid produced by an anabolic pathway catalyzed by repressible enzymes.
If tryptophan is absent, the repressor is inactive, the operon is on, and RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes.

9. Figure 18.3b – The trp Operon
As tryptophan accumulates, it inhibits its own production by activating the repressor protein.
The repressor switches the operon off by binding to the operator and blocking access of RNA polymerase to the promoter.
Tryptophan binds to an allosteric site on the protein, causing its conformation to change to the active form.
REMEMBER: The trp operon is an example of a repressible operon because it is always in the ON position until not needed – then it is switched off.

10. Figure 18.4a – The lac Operon
The lactose operon services a series of three genes involved in the process of lactose metabolism. These genes help bacteria to digest lactose.
It makes sense for these bacteria to express these genes only when lactose is present…otherwise, they waste energy on unneeded enzymes.
This is where OPERONS come in to play…the lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator on the promoter region and blocking transcription from occurring.

11. Figure 18.4b – The lac Operon
When lactose is present, there is a binding site on the repressor where lactose attaches, causing the repressor to let go of the promoter region.
RNA polymerase is then free to bind to that site and initiate transcription of the genes.
IN THIS EXAMPLE…allolactose, an isomer of lactose, de-represses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced.
REMEMBER: the lac operon is an example of an inducible operon because it is always off until it is induced to turn on.

12. Positive Gene Regulation
Prokaryotic Gene Regulation
Positive Gene Regulation
When glucose and lactose are both present in its environment, E. coli prefer to use glucose.
Only when lactose is present AND glucose is in short supply does E. coli use lactose as an energy source, and only then does it synthesize appreciable quantities of the enzymes for lactose breakdown.
How does the E. coli cell sense the glucose concentration and relay this information to its genome?

13. Figure 18.5a – Positive Control
If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA coding for the enzymes in the lactose pathway.

14. Figure 18.5b – Positive Control
When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.

15. Factors Affecting Ability of Repressor to Bind to Operator
Prokaryotic Gene Regulation
Factors Affecting Ability of Repressor to Bind to Operator
Co-Repressor : Activates a Repressor
Seen in the trp Operon
Co-Repressor is tryptophan
Turns normally “on” Operon “off”
Inducer: Inactivates a Repressor, Induces the Gene to be Transcribed
Seen in the lac Operon
Inducer is allolactose
Turns normally “off” Operon “on” 15

16. Review: Structure/Function of Prokaryotic Chromosomes
Prokaryotic Gene Regulation
Review: Structure/Function of Prokaryotic Chromosomes
shape (circular/nonlinear/loop)
less complex than eukaryotes (no histones/less elaborate structure/folding)
size (smaller size/less genetic information/fewer genes)
replication method (single origin of replication/rolling circle replication)
transcription/translation may be coupled
generally few or no introns (noncoding segments)
majority of genome expressed
operons are used for gene regulation and control
NOTE: plasmids – more common but not unique to prokaryotes/not part of prokaryote chromosome

17. The Structure of the Chromosome
Chromosome Structure
The Structure of the Chromosome
In Prokaryotes:
The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein
In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid
In Eukaryotes:
Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein
Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells
Histones are proteins that are responsible for the first level of DNA packing in chromatin

18. Chromosome Structure of Eukaryotes
Eukaryotic Chromosomes
Chromosome Structure of Eukaryotes
Most chromatin is loosely packed in the nucleus during interphase and condenses into chromosomes prior to mitosis.
Loosely packed chromatin is called euchromatin.
During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin.
Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions.
Eukaryotic chromosomes contain DNA wrapped around proteins called histones. The strands of nucleosomes are tightly coiled and supercoiled to form chromosomes.

19. Eukaryotic Chromosomes
Figure 16.21b Chromatin packing in a eukaryotic chromosome
Chromatin is organized into fibers
10-nm fiber
DNA winds around histones to form nucleosome “beads”
Nucleosomes are strung together like beads on a string by linker DNA
30-nm fiber
Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber
300-nm fiber
The 30-nm fiber forms looped domains that attach to proteins
Metaphase chromosome
The looped domains coil further
The width of a chromatid is 700 nm

20. Control of Gene Expression in Eukaryotes
Eukaryotic Gene Regulation
Control of Gene Expression in Eukaryotes
Eukaryotic gene expression can be regulated at any stage.
Because gene expression in eukaryotes involves more steps, there are more places where gene control can occur.
Opportunities for the control of gene expression in eukaryotes include:
Chromatin Packing, modification
Assembling of Transcription Factors
RNA Processing
Regulation of mRNA degradation and Control of Translation
Protein Processing and Degradation

21. Overview Figure 18.6
THIS FIGURE IS HIGHLIGHTING KEY STAGES IN THE EXPRESSION OF A PROTEIN-CODING GENE.
The expression of a given gene will not necessarily involve every stage shown.
MAIN LESSON: each stage is a potential control point where gene expression can be turned on or off, sped up, or slowed down.

22. Expression of Genes in Eukaryotes
Eukaryotic Gene Regulation
Expression of Genes in Eukaryotes
Eukaryotic cells face the same challenges as prokaryotic cells in expressing their genes, but with two main differences:
The much greater size of the typical eukaryotic genome;
importance of cell specialization in multicellular eukaryotes.
In both prokaryotes and eukaryotes, DNA associates with proteins to form chromatin, but in the eukaryotic cell, the chromatin is ordered into higher structural levels.

23. Eukaryotic Chromosome Structure
Eukaryotic Gene Regulation
Eukaryotic Chromosome Structure
Chromatin structure is based on successive levels of DNA packing.
Eukaryotic chromatin is composed mostly of DNA and histone proteins that bind to the DNA to form nucleosomes, the most basic units of DNA packing.
Additional folding leads ultimately to highly compacted heterochromatin, the form of chromatin in a metaphase chromosome.
In interphase cells, most chromatin is in a highly extended form, called euchromatin.
Chromatin structure is based on successive levels of DNA packing.
Chromatin = term for DNA + Proteins in the Nucleus of a Eukaryotic cell
DNA is wound around Histones (positively charged proteins which attract negatively charged DNA)
Histones (8) with wound DNA make up a Nucleosome - the fundamental packing unit of Chromosome (10nm fiber)
Nucleosomes further condense and form 30nm Chromatin Fibers
Nucleosomes scaffold, loop, fold to make Looped Domains (300nm – 700nm fibers)
Metaphase Chromosome – most condensed, largest form of DNA (1400nm) 23

24. Eukaryotic Gene Regulation
The Eukaryotic Genome
In prokaryotes, most of the DNA in a genome codes for protein, with a small amount of noncoding DNA that consists mainly of regulatory sequences such as promoters.
In eukaryotic genomes, most of the DNA (97% in humans) does NOT encode protein or RNA.
This DNA includes introns and repetitive DNA:
Repetitive DNA are nucleotide sequences that are present in many copies in a genome, usually not within genes.

25. Chromatin Modifications
Eukaryotic Gene Regulation
Chromatin Modifications
Chromatin modifications affect the availability of genes for transcription:
The physical state of DNA in or near a gene is important in helping control whether the gene is available for transcription.
Genes of heterochromatin (highly condensed) are usually not expressed because transcription proteins cannot reach the DNA.
DNA methylation seems to diminish transcription of that DNA.
Histone acetylation seems to loosen nucleosome structure and thereby enhance transcription.

26. Eukaryotic Gene Regulation
DNA Methylation
DNA methylation is the attachment of methyl groups (-CH3) to DNA bases after DNA is synthesized.
Methylation renders DNA inactive.
Inactive DNA, such as that of inactivated mammalian X chromosomes (Barr bodies), is generally highly methylated compared to DNA that is actively transcribed.
Comparison of the same genes in different types of tissues shows that the genes are usually more heavily methylated in cells where they are not expressed.
In addition, de-methylating certain inactive genes (removing their extra methyl groups) turns them on.
At least in some species, DNA methylation seems to be essential for the long-term inactivation of genes that occurs during cellular differentiation in the embryo.

27. Eukaryotic Gene Regulation
Histone Acetylation
Histone acetylation is the attachment of acetyl groups (-COOH3) to certain amino acids of histone proteins; de-acetylation is the removal of acetyl groups.
When the histones of nucleosome are acetylated, they change shape so that they grip the DNA less tightly.
As a result, transcription proteins have easier access to genes in the acetylated region.

28. Transcription Initiation
Eukaryotic Gene Regulation
Transcription Initiation
Transcription is controlled by the presence or absence of particular transcription factors, which bind to the DNA and affect the rate of transcription.
Thus…transcription initiation is controlled by proteins that interact with DNA and with each other.
Once a gene is “unpacked”, the initiation of transcription is the most important and universally used control point in gene expression.

29. Eukaryotic Gene and its Transcript
Figure 18.8
Eukaryotic Gene and its Transcript
REMEMBER: Transcription is controlled by the presence or absence of particular transcription factors, which bind to the DNA and affect the rate of transcription.

30. Assembling of Transcription Factors
Activator proteins bind to enhancer sequences in the DNA and help position the initiation complex on the promoter.
DNA bending brings the bound activators closer to the promoter. Other transcription factors and RNA polymerase are nearby.
Protein-binding domains on the activators attach to certain transcription factors and help them form an active transcription initiation complex on the promoter.
Control elements are simply segments of noncoding DNA that help regulate transcription of a gene by binding proteins (transcription factors). 30

31. Post-Transcriptional Factors
Eukaryotic Gene Regulation
Post-Transcriptional Factors
Transcription alone DOES NOT constitute gene expression!
Post-transcriptional mechanisms play supporting roles in the control of gene expression:
Alternative RNA splicing – where different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns.
Regulatory proteins specific to a cell type control intron-exon choices by binding to regulatory sequences within the primary transcript.

32. Alternative Splicing Offers New Combinations of Exons = New Proteins
Eukaryotic Gene Regulation
Alternative Splicing Offers New Combinations of Exons = New Proteins
The RNA transcripts of some genes can be spliced in more than one way, generating different mRNA molecules.
With alternative splicing, an organism can get more than one type of polypeptide from a single gene. 32

33. Further Control of Gene Expression
Eukaryotic Gene Regulation
Further Control of Gene Expression
After RNA processing, other stages of gene expression that the cell may regulate are mRNA degradation, translation initiation, and protein processing and degradation.
The life span of mRNA molecules in the cytoplasm is an important factor in determining the pattern of protein synthesis in a cell.
Most translational control mechanisms block the initiation stage of polypeptide synthesis, when ribosomal subunits and the initiator tRNA attach to an mRNA.

34. Protein Processing and Degradation
Eukaryotic Gene Regulation
Protein Processing and Degradation
The final opportunities for controlling gene expression occur after translation:
Protein processing – cleavage and the addition of chemical groups required for function.
Transport of the polypeptide to targeted destinations in the cell.
Cells can also limit the lifetimes of normal proteins by selective degradation – chopped up by proteasomes.

35. Overview Figure 18.6
THIS FIGURE IS HIGHLIGHTING KEY STAGES IN THE EXPRESSION OF A PROTEIN-CODING GENE.
The expression of a given gene will not necessarily involve every stage shown.
MAIN LESSON: each stage is a potential control point where gene expression can be turned on or off, sped up, or slowed down.

36. The Molecular Biology of Cancer
The Biology of Cancer
The Molecular Biology of Cancer
Certain genes normally regulate growth and division – the cell cycle – and mutations that alter those genes in somatic cells can lead to cancer.
Proto-Oncogenes are normal genes that code for proteins which stimulate normal cell growth and division.
Oncogenes – cancer causing genes; lead to abnormal stimulation of cell cycle. Oncogenes arise from genetic changes in proto-oncogenes:
Amplification of proto-oncogenes
Point mutation in proto-oncogene
Movement of DNA within genome

37. The Biology of Cancer
Genetic Changes Can Turn Proto-oncogenes into Oncogenes
Proto-oncogenes can be converted to oncogenes by
Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase
Amplification of a proto-oncogene: increases the number of copies of the gene
Point mutations in the proto-oncogene or its control elements: causes an increase in gene expression 37

38. Tumor-Suppressor Genes
The Biology of Cancer
Tumor-Suppressor Genes
In addition to mutations affecting growth-stimulating proteins, changes in genes whose normal products INHIBIT cell division also contribute to cancer:
Such genes are called tumor-suppressor genes because the proteins they encode normally help prevent uncontrolled cell growth.

39. The Biology of Cancer
p53 Tumor Suppressor and ras Proto-Oncogenes
Mutations in the p53 tumor-suppressor gene and the ras proto-oncogene are very common in human cancers.
Both are components of signal-transduction pathways that convey external signal to the DNA in the cell’s nucleus.
Product of ras gene is G Protein (relays a growth signal and stimulates cell cycle).
An oncogene protein that is a hyperactive version of this protein in the pathway can increase cell division.
P53 protein – “guardian angel of the genome”
DNA damage (UV, toxins) signals expression of p53 and p53 protein acts as transcription factor for gene p21
p21 halts cell cycle, allowing DNA repair
P53 also can cause ‘cell suicide’ if damage is too great
Many cancer patients p53 gene product does not function properly!

40. Figure 18.21 Signaling pathways that regulate cell growth (Layer 2)
RAS and P53 contribute to uninhibited cell stimulation and growth- Tumor Formation
Figure Signaling pathways that regulate cell growth (Layer 2) 40

41. The Biology of Cancer
Figure A multi-step model for the development of colorectal cancer 41

42. Review: Structure/Function of Eukaryotic Chromosomes
Chromatids
2/sister/pari/identical DNA/ genetic information
distribution of one copy to each new cell
Centromere
noncoding/uncoiled/narrow/constricted region
joins/holds/attaches chromatids together
Nucelosome
histones/DNA wrapped arround special proteins
packaging compacting
Chromatin Form (heterochromatin/euchromatin)
heterochromatin is condensed/supercoiled
proper distribution in cell division (not during replication)
euchromatin is loosely coiled
gene expression during interphase/replication occurs when loosely packed
Kinetochores
disc-shaped proteins
spindle attachment/alignment
Genes or DNA
brief DNA description
codes for proteins or for RNA
Telomeres
tips, ends, noncoding repetitive sequences
protection against degradation/ aging, limits number of cell divisions

43. USEFUL ANIMATIONS

44. You should now be able to:
NEED TO KNOW
You should now be able to:
Explain the concept of an operon and the function of the operator, repressor, and corepressor
Explain the adaptive advantage of grouping bacterial genes into an operon
Explain how repressible and inducible operons differ and how those differences reflect differences in the pathways they control

45. Define control elements and explain how they influence transcription
NEED TO KNOW
Explain how DNA methylation and histone acetylation affect chromatin structure and the regulation of transcription
Define control elements and explain how they influence transcription
Explain the role of promoters, enhancers, activators, and repressors in transcription control

46. Explain how eukaryotic genes can be coordinately expressed
NEED TO KNOW
Explain how eukaryotic genes can be coordinately expressed
Describe the roles played by small RNAs on gene expression
Explain why determination precedes differentiation
Describe two sources of information that instruct a cell to express genes at the appropriate time

47. Describe the effects of mutations to the p53 and ras genes
NEED TO KNOW
Explain how mutations in tumor-suppressor genes can contribute to cancer
Describe the effects of mutations to the p53 and ras genes