1. Control of Prokaryotic
Gene Expression

2. Prokaryotic Regulation of Genes
Regulating Biochemical Pathway for Tryptophan Synthesis.
Produce something that will interfere with the function of the enzyme in the pathway.
Produce a gene regulator that can inhibit the transcription of one biochemical pathway enzymes.
In all cells, there are genes that code for proteins. It would be wasteful if genes were expressed when there is not a need for the protein product.
Example - why make enzymes used to break glycogen into glucose if the cell has a reservoir of glucose molecules? OR if the cell has no glycogen?
Regulating Prokaryotic Genes
Prokaryotic regulation is different from eukaryotic regulation.
Prokaryotic gene regulation tends to be negative. Meaning that the gene is usually activated unless some regulator inhibits it or deactivates it.
Eukaryotic gene regulation is usually positive. Meaning that the gene is usually deactivated unless a regulator activates it or turns it “on”.

3. Eukaryotic cells have many more genes (i. e
Eukaryotic cells have many more genes (i.e. 23,000 in human cells) in their genomes than prokaryotic cells (i.e. average 3000).
Physically there are more obstacles to regulate eukaryotic genes because there is so much more DNA to manage. For example, eukaryotic chromatin is wrapped around histone proteins.
In addition there are other nonhistone proteins that are used in eukaryotic gene expression that are not used in prokaryotic gene expression.

4. Operon and Prokaryotic Gene Expression
Operon- A group of prokaryotic genes with a related function that are often grouped and transcribed together. In addition, the operon has only one promoter region for the entire operon. PROG

5. Operon and Prokaryotic Gene Expression
PROG
An operon is composed of the following:
Structural genes- genes that are related and used in a biochemical pathway.
Promoter-The nucleotide sequence that can bind with RNA polymerase to start transcription. This sequence also contains the operator region.
Operator-The nucleotide sequence that can bind with repressor protein to inhibit transcription.
Initiation begins when RNA polymerase attaches to the promoter region – a recognition site that contains a group of nucleotides TATA (tata box)
As in DNA replication, the polymerase of transcription needs the assistance of helper proteins to find attach to the promoter region. These helpers are called transcription factors
RNA processing

6. Regulator Genes and Repressors
Regulator gene- This gene produces a protein called a repressor that can inhibit the transcription of an operon by attaching to the operator (upstream or downstream).

7. Interaction of Modulators and Repressors
Repressors have allosteric properties. Modulators can bind to the repressor at an allosteric site changing the conformation of the repressor, thereby activating or deactivating the repressor. Usually the modulator is a product of the biochemical pathway.
Prokaryotic Gene Expression
Prokaryotic genes with related function are often grouped together. This grouping is called an operon. All the related genes will be transcribed together. In addition, the operon has only one promoter region for the entire operon. An operon is composed of the following:
-Structural genes- genes that are related and used in a biochemical pathway.
-Regulator gene- This gene will produce a repressor protein that will turn the operon off or inhibit the transcription of the operon. Repressors have allosteric properties. Modulators can bind to the repressor at an allosteric site changing the conformation of the protein. The modulator is usually a product of the biochemical pathway.

8. Lactose and the Inducible lac Operon
Negative Gene Regulation
1. Inducible operon- the lac operon. This operon has the ability to convert lactose into glucose and galactose. This involves three structural genes
Inducible Operon-Is an operon that is induced, or turned on, by a particular modulator that inhibits the repressor. Inhibiting the repressor will allow for the transcription of the operon. i.e. inducible operons are “off” unless something turns them “on” – inducing them to act.
Example lac operon
The products of the lac operon produces enzymes necessary to break down lactose into glucose and galactose. This allows the prokaryote to use the disaccharide lactose as an energy source when necessary. There is no need to use this gene unless lactose is present, so the gene is “off” unless lactose induces it to be “on.”
Three structural genes of the lac operon
lacZ-makes b-galactosidase an enzyme that breaks lactose into glucose and galactose
lacY-makes permease which increases the cell's permeability for lactose.
lacA-makes transacetylase whose function is unknown.
The lac operon is an example of an inducible operon.

9. Animation of the lac Operon and Presence of Lactose

10. Absence of Lactose and the lac Operon
If no lactose or allolactose is present, the repressor protein is active, binding to the operator site. This prohibits the RNA polymerase from transcribing the operon.

11. Animation of the lac Operon and Absence of Lactose

12. Synthesis of Tryptophan and the Repressible trp Operon
The purpose of the trp operon is to synthesize the amino acid tryptophan when needed.
Unlike inducible operons, repressible operons are “on” unless the presence of something actively turns them “off” – represses them to make them inactive.
Repressible operon-the trp operon. This is a repressible operon because it is “on” unless excess tryptophan is present. When the cell does not have tryptophan, the repressor enzyme is inactive. RNA polymerase can attach to the promoter region and transcribe the genes necessary for the synthesis of tryptophan. When tryptophan is present, however, it represses the operon or inhibits the transcription of the operon it by activating the repressor protein. The trp operon includes five structural genes that make five enzymes necessary for the synthesis of tryptophan. The repressor protein that controls this operon is inactive without tryptophan present. Tryptophan is called a corepressor as it attaches to the repressor.

13. Animation of the trp Operon and Absence of Tryptophan

14. Tryptophan Present and the Repressible trp Operon
The repressor protein becomes active when tryptophan is present. The repressor protein binds to the operator region blocking RNA polymerase from attaching to the promoter region.

15. Animation of the trp Operon and Presence of Tryptophan

16. Lac and trp Operons-Examples of Negative Gene Regulation
The lac and typ operons are example of negative gene regulation as the repressor protein inhibits transcription of the operons.

17. Example of Positive Gene Regulation
While most prokaryotic gene regulation is negative, there are some examples of positive gene regulation. The lac operon has both a negative and a positive way to regulate the gene. The purpose of the lac operon is to break down lactose when lactose is present and when glucose is absent. The way the cell senses whether or not it has glucose involves another regulatory gene. This produces cAMP receptor protein (CRP). The CRP binds to a site next to the promoter region making it easier for the RNA polymerase to attach. The CRP is an allosteric protein. It becomes active when cAMP binds to it. cAMP accumulates in the cell when glucose is absent.

18. Both Lactose and Glucose Present
Lactose present, glucose present (cAMP level low), little lac mRNA synthesized

19. Control of Eukaryotic
Gene Expression

20. Eukaryotic Gene Regulation
Prokaryotic regulation is different from eukaryotic regulation.
Eukaryotic cells have many more genes (23,700 in human cells) in their genomes than prokaryotic cells (average 3000).
Physically there are more obstacles as eukaryotic chromatin is wrapped around histone proteins.
There are more non-histone proteins that are used in eukaryotic gene expression than in prokaryotic gene expression.
Current estimates from the Human Genome Project show that there are about 23,700 protein-coding genes in the human genome; BUT they are responsible for producing about 160,000 different proteins. This is possible due to alternative splicing during RNA Processing. Each protein coding gene has an average of 7 different products. 20 20

21. Eukaryotic Gene Regulation in Multicellular Organisms
Almost all the cells in an organism are genetically identical or totipotent.
Differences between cell types result from differential gene expression -- the expression of different genes by cells with the same genome.
Errors in gene expression can lead to diseases including cancer.
Gene expression is regulated at many stages.
“Totipotent” = a cell with a full set of genes; i.e. has the total potential to become any type of cell.
White blood cells (lymphocytes) are cells that are not totipotent and are different from one another in the genes that produce antibodies.

22. Organization of DNA http://www.youtube.com/watch?v=gbSIBhFwQ4s
Organization of the genome in eukaryotic cells has an impact on gene regulation. The genome is organized with the help of proteins. The DNA is double stranded and forms a helix. That helix then is wound around 8 histone proteins g (2 copies of 4 different histone proteins) forming beads called nucleosomes. There is a fifth histone protein (H1) that binds to the DNA outside the nucleosome. There are five different histone proteins, H2A, H2B, H3, and H4. The histones remain attached to the DNA except when DNA is replicating. Even when the DNA is being transcribed, the histones remain attached. Histones have the ability to change position and shape to allow RNA polymerases to transcribe the DNA gene.
Double stranded DNA is wrapped around histone proteins like beads on a string.
With the help with H1 histone protein, the nucleosomes coil to form chromatin fibers of 30 nm in diameter.
At the next level are looped domains that are connected to a nonhistone protein scaffold.
The chromatin folds further for a maximum compacted chromosome.
During interphase, (G1, S, G2), the DNA uncoils to the looped domain level. Certain looped domains are restricted to certain areas of the nucleus.
Approximately 97% of the DNA found in a human cell is not used for protein-coding genes. Each specialized cell activates a variety of genes.

23. Types of Repetitive DNA
Types of noncoding DNA
Introns-intervening sequences found in DNA gene. An average human gene is 36,000 base pairs (bp) long, but contains 6 introns that are about 5700 bp each and 7 exons that are about 300 bp each. So, the actual coding region is only 2100 bp for a 36,000 bp gene. (a bit of trivia: by bp number, if we include the introns, 25% of your genome is composed of protein-coding genes, but if we only count the exons, then only 1.3% of your genome codes for protein)
Repetitive DNA is found between genes-Two types
Tandemly Repetitive DNA or satellite DNA. Certain sequences of base pairs are repeated over and over again sequentially. For example- The sequence GTTAC is repeated as shown GTTACGTTACGTTACGTTACGTTACGTTAC.
Tandemly repetitive DNA can be found at the ends of the chromosomes called telomeres. The lagging side of chromosome will shorter. To compensate for shortening telomeres, telemerase will restore the missing repetitive sequences.
Interspersed repetitive DNA
These are larger segments of DNA that are repeated throughout the DNA genome. There may be slightly different from one repeat to another.

24. Altering the Genome is a Form of Gene Regulation
Gene amplification- In amphibian ovum there are over 1 million copies of the rRNA made from tiny circles of DNA in the nucleoli.
Gene Loss-In gall midges (an insect) during development, all but two cells lose 32 of their 40 chromosomes during the first mitotic divisions. These cells that retain 40 chromosomes will produce gametes.
Transposed genes-The hemoglobin gene has been duplicated, mutated, and transposed to other chromosomes to produce multiple but different copies of the same gene.

25. Gene Duplication and Transposition Regulates Genes
Genes are usually not created from scratch. They are often duplicated and moved from one area to another. Each gene will experience different mutations. This will give rise to different proteins. Quite often these are called gene families. The illustration above is the evolution of the globin family.

26. Rearrangement Gene Domains Regulates Genes
B lymphocytes can produce millions of different types of antibodies (proteins) that react with millions of different antigens. This happens by rearrangement of AB genes.
An antibody (AB) has four polypeptide chains, each with a constant region and with a variable region (V). The antibody gene has hundreds of variable regions which are adjacent to the junction region and constant region. During differentiation, parts of the region V rearrange themselves to produce unique antibodies for different antigens.

27. Epigenetics
Epigenetics refers to processes that influence gene expression or function without changing the underlying DNA sequence.
Acetylation
Methylation

28. Acetylation
Acetylation of lysine found on the histone decreases the affinity of histones for DNA and other histones, thereby making DNA more accessible for transcription.

29. Methylation
A methyl group can be added to the nitrogenous bases of cytosine that are followed by guanine. Many human genes have upstream CG-rich regions called CpG islands. Methylation of a gene's CpG island represses gene expression. Different cells have different methylation patterns, which contributes to the differences in gene expression in different cell types.
The Barr body of the second X chromosome in females is highly methylated. Once a gene is methylated, it stays methylated even when the cell divides. The daughter cells will keep the genes methylated. This is called genomic imprinting and the gene remains inactive.

30. Role of Transcription Factors
The role of enhancers and transcription factors.
a. Transcription factors bind to the promoter region (TATA box) and must be present for the RNA polymerase to attach.
b. Upstream from the promoter region there may be an inducer region which may help transcription factors to bind. These are called proximal control elements
c. In addition, there may be a DNA site distal control elements even further upstream (thousands of nucleotides away) called a enhancers. DNA enhancers can work by a protein (activator) attaching to the enhancer. The DNA then loops the DNA back on itself to attach to the promoter region. When this happens, it is easier for the transcription factors and RNA polymerase to attach so that transcription can occur.

31. Role of Transcription Factors
The enhancers attract activators. These activators and the region of the DNA they are attached to are attracted to the promoter region of the DNA gene. It causes actual folding of the DNA gene which, in turn, attracts more transcription factors and the attachment of RNA polymerase.

32. Role of Transcription Factors and Activators
This illustrates how different cells have different activators which activate different genes.
The liver cells need the protein albumin and not the protein crystallin and the lens cells do not need albumin but do need crystallin.
Note how each cell has a unique set of activators and transcription factors that interact with only certain genes in the cell.

33. Role of Transcription Factors and Lactose Persistence
The LCT gene produces the enzyme lactase which digests lactose. Lactose is a disaccharide found in dairy products. In order to be transcribed, the LCT gene needs a regulatory protein coded for by the MCM6 gene. Most humans after weaning cease to produce the regulatory protein but a mutation in the gene will allow its continued production through adulthood. This mutation causes a condition called lactose persistence.
The role of enhancers and transcription factors.
A regulatory gene called MCM6 is responsible for the decline in the expression of LCT gene once a person is weaned. If the LCT gene is not transcribed, then lactase will not be produced and the person is said to be lactose intolerant (2/3 of all humans are lactose intolerant). For a very interesting short video on the subject of lactose tolerance vs intolerance and its evolution in humans see
A specific DNA sequence within the MCM6 gene, called a regulatory element, helps control the activity (expression) of a nearby gene called LCT. The LCT gene provides instructions for making an enzyme called lactase. This enzyme helps to digest lactose, a sugar found in milk and other dairy products. Lactose intolerance in adulthood, which occurs in most humans, is caused by gradually decreasing expression of the LCT gene after infancy,. Mutations in the MCM6 gene allow the LCT gene to continue to be transcribed. The mechanisms of how the MCM6 gene causes the eventual decline in the transcription of the LCT are still being investigated.
At least four variations have been identified in the regulatory element that modulates LCT gene expression. These variations change single DNA building block (nucleotides) in the regulatory element. Each of the mutations results in sustained lactase production in the small intestine and the ability to digest lactose throughout life. People without these changes have a reduced ability to digest lactose as they get older, resulting in the signs and symptoms of lactose intolerance.
The LCT gene is located on chromosome #2 at 136,545,414 to 136,594,749. The MCM6 gene is located upstream at 136,597,195 to 136,634,046

34. Coordination of Expression of Related Proteins in a Biochemical Pathway
Quite often related genes needed for a given biochemical pathway are found on different chromosomes, yet they need to be transcribed and translated at the same time. This happens because the related genes use the same activator protein or transcription factors.
For example, sex hormones have multiple effects and turn on a number of related genes. This is because the steroid hormone has the ability to combine with a receptor protein when it enters the cell. This becomes a transcription factor that activates certain genes and not others. The sex hormone may activate more than one receptor protein once inside the cell.

35. Post-Transcriptional Control Alternative Splicing
Once the immature mRNA is made, it could be processed in different ways to give rise to different mature mRNA and thus different proteins.
Fot example,. In Drosophila there is a gene that is called a "double--sex" gene because the mRNA can be processed two different ways depending on the presence of a tra gene product. The "double-sex" gene has 6 exons and in females the tra gene product is present. It causes exon 3 to be linked to exon 4 but the remaining exons are deleted. In males the tra gene product is absent. This causes exon 3 to be linked to 5 and 6, skipping # 4 altogether. Once the mRNA is made, it will lead to development of either a male or female.

36. Second Example
In Drosophilia there is a gene called Tropin T. There are enough exons and introns in the gene that the mRNA from this gene can be spliced over 17,000 ways giving rise to 17,000 different proteins. These unique proteins are found on different nerve cells in Drosophilia.

37. Example of Alternative Splicing the Same Gene in Humans
Current estimates from the Human Genome Project show that there are about 23,700 protein-coding genes in the human genome; BUT they are responsible for producing about 160,000 different proteins. This is possible due to alternative splicing during RNA Processing. Each protein coding gene has an average of 7 different products. 95% of human genes undergo alternative splicing.
An average human gene is 36,000 base pairs (bp) long, but contains 6 introns that are about 5700 bp each and 7 exons that are about 300 bp each. So, the actual coding region is only 2100 bp for a 36,000 bp gene. (a bit of trivia: by bp number, if we include the introns, 25% of your genome is composed of protein-coding genes, but if we only count the exons, then only 1.3% of your genome codes for protein)
CGRP (calcitonin gene related peptide) is a hormone that 37 is amino acids long and produced in the hypothalamus. This molecule interacts with receptors in nervous tissue. It is a potent peptide vasodilator and can function in the transmission of pain. It is thought to be a part of the cause of migraine headaches.
Calcitonin which is produced by the thyroid gland is formed from the alternative splicing of the same gene as CGRP (calcitonin/CGRP gene). This located on chromosome 11. Calcitonin increases the concentration of calcium in the blood when secreted from the thyroid gland.

38. Post-Transcriptional Control and RNAi Alternative
mRNA molecules do not remain functional indefinitely. This length of time can affect the number of protein product synthesize.
Small pieces of RNA can interfere (RNAi) with mRNA by being complementary to a small part of the mRNA and tagging it for destruction.
RNA interference — or RNAi — is a cellular process that can silence specific mRNA: that is, it can stop proteins being produced from specific mRNA. RNAi can involve different types of small RNA molecules. The best known types of RNAi act by targeting messenger RNA (mRNA) in the cytoplasm. The two most known commonly known are “silence RNA” or “small interfering RNA” (siRNA) and microRNA (miRNA).
miRNA is most often transcribed from noncoding regions of the DNA. Quite often on the longer piece of DNA there are regions that are complementary to itself (A-U) and (G-C). This allows the RNA to fold back on itself in the nucleus. A protein complex with RNAase activities called Drosha clips dangling pieces of the RNA from the hair pin loop.
It attached to another protein called exportin and to exits the nucleus.
Once miRNA is in the nucleus, a protein called dicer clips the hairpin loops so that it becomes truly double stranded and is called mature miRNA.
The miRNA attaches to another protein complex called Argonaute. When the miRNA and AGO join, one side of the miRNA disengages making the RNA single stranded. This is now called RISC (RNA induced silencing complex) complex and starts “frisking” mRNA.
If there is a complementary base pairing between RISC and a given mRNA, then translation is halted. The target mRNA is then degraded.

39. Post-Transcriptional Control and microRNA (miRNA)
This is like the previous slide but in more detail.

40. Post-transcriptional control- siRNA
Another type of RNAi is siRNA or silence RNA (also called small interfering RNA). This ds RNA can come from the environment, or be transcribed from a transposon, or a virus.
The double stranded RNA is clipped by dicer repeatedly. The miRNA attaches to another protein complex called Argonaute. When the siRNA and AGO join, one side of the siRNA disengages making the RNA single stranded. This is now called RISC complex and starts “frisking” mRNA.
If there is a complementary base pairing between RISC and a given mRNA, then translation is halted. The target mRNA is then degraded.
The power of RNAi to silence specific genes is now widely used in the laboratory to explore the functions of genes. A popular approach is to use 'hairpin' RNAs (RNA molecules that fold back on themselves so that they become double-stranded). Specific siRNAs are now available to silence almost any gene in human cells or model organisms. Researchers hope to use siRNAs to correct faulty gene expression in humans. The delivery method is an important consideration for the development of RNAi-based therapies; the siRNA trigger needs to be delivered efficiently and may need to be targeted to a specific tissue.
The difference between miRNA and siRNA is a matter of their origin. miRNA origins comes from a DNA template in genome of the cell. siRNA can come from dsRNA virus, or exogenous RNA from the environment or man made DNA.

41. Other Factors that Can Affect the Expression of Genes-Post Transcription
Chemical signals that regulate the mRNA leaving the nucleus. Nearly half of all mature mRNA never reaches the cytoplasm. There must be some sort of inhibitor that will allow certain mRNA to leave and others toremain.
Degrading of the mRNA that affects its lifespan. The life-span can be associated with the length of the poly-A-tail. As it shortens, it aids other enzymes with the removal of the 5'cap and nucleases break down the mRNA. mRNA can last from minutes to weeks.

42. Post-translational control- Ubiquitin
Proteins that need to be degraded are tagged with a protein called ubiquitin. Once tagged, a protein complex called a proteasome breaks down the protein. The ubiquitin molecules are recycled and the small polypeptide fragments are further degraded by other enzymes.

43. Other Factors that Can Affect the Expression of Genes- Post Translation
Post-translational control
Modification of protein product. Often amino acids must be removed in order for the protein to be functional.
Proteins are often modified with prosthetic groups to make them functional.
Examples of Post Translational Control of Gene Expression
1. All eukaryotic proteins begin with methionine (start), yet most functional proteins no longer have this beginning amino acid. It has been removed.
2. Most digestive enzymes are made in a pre-cursor form called zymogens. This is to prevent the enzymes from digesting the cell itself. Once it is excreted, it is modified to become functional.
Examples of adding prosthetic group.
1. Hemoglobin adds a heme group to each of its polypeptide chains. If this group is in short supply, a regulatory protein inactivates an essential translating initiation factor halting all translation.