Gene Regulation

1. Gene Regulation

2. Essential knowledge 2.C.1: • Organisms use feedback mechanisms to maintain their internal environments and respond to external environmental changes. • c.Alteration in the mechanisms of feedback often results in deleterious consequences.

3. Essential knowledge 3.B.1: • Gene regulation results in differential gene expression, leading to cell specialization. • a. Both DNA regulatory sequences, regulatory genes, and small regulatory RNAs are involved in gene expression. • b. Both positive and negative control mechanisms regulate gene expression in bacteria and viruses. • c. In eukaryotes, gene expression is complex and control involves regulatory genes, regulatory elements and transcription factors that act in concert. • d. Gene regulation accounts for some of the phenotypic differences between organisms with similar genes.

4. Essential knowledge 3.B.2: • A variety of intercellular and intracellular signal transmissions mediate gene expression. • a. Signal transmission within and between cells mediates gene expression. • b. Signal transmission within and between cells mediates cell function.

5. Essential knowledge 2.C.1: • Organisms use feedback mechanisms to maintain their internal environments and respond to external environmental changes. • a. Negative feedback mechanisms maintain dynamic homeostasis for a particular condition (variable) by regulating physiological processes, returning the changing condition back to its target set point. • b. Positive feedback mechanisms amplify responses and processes in biological organisms. The variable initiating the response is moved farther away from the initial set-point. Amplification occurs when the stimulus is further activated which, in turn, initiates an additional response that produces system change.

6. Essential knowledge 2.E.1: • Timing and coordination of specific events are necessary for the normal development of an organism, and these events are regulated by a variety of mechanisms. • a. Observable cell differentiation results from the expression of genes for tissue-specific proteins. • b. Induction of transcription factors during development results in sequential gene expression. • c. Programmed cell death (apoptosis) plays a role in the normal development and differentiation.

7. Essential knowledge 3.C.1: • Changes in genotype can result in changes in phenotype. • a. Alterations in a DNA sequence can lead to changes in the type or amount of the protein produced and the consequent phenotype. • b. Errors in DNA replication or DNA repair mechanisms, and external factors, including radiation and reactive chemicals, can cause random changes, e.g., mutations in the DNA. • c. Errors in mitosis or meiosis can result in changes in phenotype.

8. Essential knowledge 3.C.1: • Changes in genotype can result in changes in phenotype. • Changes in genotype may affect phenotypes that are subject to natural selection. Genetic changes that enhance survival and reproduction can be selected by environmental conditions.

9. Essential knowledge 3.D.1: • Cell communication processes share common features that reflect a shared evolutionary history. • a. Communication involves transduction of stimulatory or inhibitory signals from other cells, organisms or the environment. • b. Correct and appropriate signal transduction processes are generally under strong selective pressure. • c. In single-celled organisms, signal transduction pathways influence how the cell responds to its environment. • d. In multicellular organisms, signal transduction pathways coordinate the activities within individual cells that support the function of the organism as a whole.

10. Essential knowledge 4.C.2: • Environmental factors influence the expression of the genotype in an organism. • a. Environmental factors influence many traits both directly and indirectly. • b. An organism’s adaptation to the local environment reflects a flexible response of its genome.

11. Gene Regulation in Prokaryotes • Gene expression is the overall process by which genetic information flows from genes to proteins • The main way gene expression is regulated is by turning transcription on or off

12. The lac operon • When lactose is plentiful in our intestine, E.colibacteria make the enzyme lactase, which helps breakdown lactose -

13. Jacob and Monod proposed a hypothesis that explained how E.coli could adjust enzyme production in response to whether or not lactose was present • E.coli require three enzymes to take up and metabolize lactose-these three genes are regulated together as a unit, and are located next to each other on the bacterial chromosome

14. In addition to the three genes, two control sequences are located adjacent to these genes • These are the promoter and the operator sequences • The operator region located after the promoter acts as the on-off switch by determining whether RNA polymerase can attach and transcribe genes • The combined promoter, operator and structural genes are collectively called the operon. Operons only occur in prokaryotes

15. Negative control of the lac operon by repressors • A regulatory gene located near the operon produces a repressor protein • The repressor protein can bind to the operatorregion, which prevents RNA polymerase from binding to the promoter region - this blocks transcription

16. When lactose is present, the enzymes used for its breakdown are needed • When lactose is present, lactose binds to the repressor which changes its shape causing it to become inactive, this prevents it from binding to the operator region • This allows RNA polymerase to bind to the promoter region so that transcription can occur, and the enzymes needed are produced

17. The lac mRNA and enzymes breakdown quickly, so when lactose is no longer present, the repressor again becomes active and binds to the operator, preventing transcription • The lac operon is called an inducible operon because when lactose binds to the repressor, the repressor is inactivated - this induces transcription

18. Positive control of the lac operon by catabolite activator protein (CAP) • The control of the lac operon is also regulated by a positive control mechanism • Glucose is the preferred source of energy in bacteria. If glucose levels are low, breakdown of lactose as an energy source is necessary • cAMP binds to a regulatory protein called CAP, which activates this protein

19. The CAP protein binds to a CAP binding site located next to the promoter region • This helps the RNA polymerase to bind to the promoter region which allows transcription to occur much faster (20-50X) • CAP is an transcription activator

20. When lactose and glucose are present, the bacteria will use glucose as its preferred energy source, so they need very little of the enzymes to break down lactose • This results in CAP not being activated • Since CAP is not activated, RNA ploymerase binds poorly to the promoter and transcription of the lactose enzymes is very slow, and very little mRNA is made

21. The trp (tryptophan)operon • In the trp operon, the repressor is normally inactive, so transcription occurs without being hindered • When tryptophan is present, it binds to the repressor making the repressor active, the active repressor binds to the operator region and blocks transcription • Tryptophan is acting as a corepressor

22. Tryptophan binds to the repressor which is then activated, the active repressor binds to operator - this represses transcription • This allows the bacteria to stop making a certain essential molecule such as tryptophan when it is already present in sufficient amounts, because the active repressor binds to operator and stops transcription

23. Comparing the lac operon and the trp operon • The trp operon is a repressible operon • Involved in pathways that synthesize a substance neededby the cell • Repressor normally inactive so that substance will be made • If desired substance is present in medium, or in high concentrations in the cell, it is not needed so the substance itself (tryptophan) acts as a corepressor (activates repressor) and turns off transcription • The lac operon is an inducible operon • Involved in pathways that breakdown a nutrient – enzyme is needed only when nutrient is present • Repressor is normally active because enzyme is not needed

24. Activators are proteins that bind to DNA at the enhancer region • This helps RNA polymerase to bind to the promoter region

25. Every somatic cell has a copy of every gene • What type of cell a cell is depends on what genes are turned on and what genes are turned off • The regulation of genes therefore leads to specialization

26. The zygote has a complete set of genes for making all the types of specialized cells an organism will need • Differentiation does not necessarily involve irreversible changes in the DNA….specialized cells retain the potential to become any kind of cell • The differences among cells in an organism result from the selective expression of genes

27. In both prokaryotes and eukaryotes, initiation of transcription is the most important stage for regulating gene expression • Eukaryotic cells contain more regulatory proteins and more control sequences in their DNA

28. Prokaryotes use repressors and activators • Eukaryotes use both, but activator proteins seem to be more important than repressors are • The default (normal) state for most genes is off • Turning on eukaryotic genes involves regulatory proteins called transcription factors

29. Activators are a type of transcription factor • Basal proteins (general transcription factors) bind to TATA box on promoter to form transcription factor complex • Other proteins (mediator) link transcription factors to activators • This complex interaction of activator proteins helps RNA polymerase to bind to promoter region and begin transcription more efficiently

30. An activator is a protein that binds to an enhancer region located far downstream of the gene, and stimulates transcription of a gene

31. Eukaryotic Gene Regulation • Gene regulation also occurs during other processes besides transcription • Processing of mRNA primary transcript must occur before translation (cap, poly A tail, exon splicing, export) • mRNA breakdown - mRNA can last from a few hours in prokaryotes to hours, even weeks in eukaryotes

32. Translation - many regulatory proteins needed for translation • Post-translational - many proteins do not become active until they are edited, other proteins breakdown quickly

33. Eukaryotic Regulation • A variety of mechanisms • Five primary levels of control: • Nuclear levels • Chromatin Packing • Transcriptional Control • Posttranscriptional Control • Cytoplasmic levels • Translational Control

34. Chromatin Structure • Eukaryotic DNA associated with histone proteins • Together make up chromatin • As seen in the interphase nucleus • Nucleosomes: • DNA wound around balls of eight molecules of histone proteins • Looks like beads on a string • Each bead a nucleosome • The levels of chromatin packing determined by degree of nucleosome coiling

35. Chromatin Packing • Euchromatin • Loosely coiled DNA • Heterochromatin • Tightly packed DNA • Barr Bodies • Females have two X chromosomes, but only one is active • Other is tightly packed along its entire length • Inactive X chromosome is Barr body

36. Transcriptional Control • Transcription controlled by proteins called transcription factors - TF’s bind to promoter region and bind RNA polymerase • Transcription activators bind to enhancer regions on DNA, regions of DNA where factors that regulate transcription can also bind

37. Transposons (jumping genes) • Discovered by Barbara McClintock • Specific DNA sequences that move within and between chromosomes • Their movement may alter neighboring genes, decreasing their expression-act like regulator genes

38. Posttranscriptional Control • Posttranscriptional control operates on primary mRNA transcript • Given a specific primary transcript: • Excision of introns can vary • Splicing of exons can vary • Determines the type of mature transcript that leaves the nucleus • May also control speed of mRNA transport from nucleus to cytoplasm • Will affect the number of transcripts arriving at rough ER

39. Translational Control • Translational Control - Presence or absence of 5′ cap and poly-A tail on 3′ end determines whether mRNA is translated into a protein product, and how long mRNA is active • Posttranslational Control - Affects the activity of a protein product • Activation of proteins after being translated (enzymes = zymogens)

40. In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellularphenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- (Greek: επί- over, above, outer) -genetics. • It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. • .

41. Actively transcribed genes are associated with accessible chromatin regions, while transcriptionally silent genes are often in inaccessible chromatin regions. • The modifications made to DNA and proteins that impact chromatin structure are referred to as epigenetic markers (or marks). Examination of histone acetylation patterns has demonstrated a high correlation between histone acetylation and active transcription, whereas histone methylation can be associated with the activation or silencing of genes depending on the amino acid modified and the number of methyl groups added.

42. Histones order DNA into structural units called nucleosomes. Histones are the major protein component of chromatin and are subject to several different post-translational modifications, including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation. • Acetylation of histones typically leads to relaxed chromatin and is associated with activation of transcription. In contrast, histone deacetylases (HDACs) catalyze the removal of acetyl groups from histones, consequently leading to more tightly packaged chromatin. • DNA methylation often leads to the compaction of chromatin via the recruitment of enzymes that methylate histones, creating an additional level of transcriptional repression.

43. Eukaryotic genome • In general, more complex organisms have more complex genes with more and larger introns • In humans 95% or more of average protein-coding gene is introns • Introns allow exons to be put together in various combinations, producing different proteins from a single gene, may also regulate gene expression and help to determine which genes are to be expressed • Humans have ~6 billion base pairs in 2N human genome • ~23-25,000 genes • Protein-coding genes make up only about 1.5 - 2 % of our total DNA • Typical human cell expresses only 3 – 5% of its genes at a given time • DNA sequences between genes are called intergenic sequences

44. Transduction pathways may regulate genes by activating transcription factors that turn genes on or off, thereby regulating the synthesis of enzymes

45. Steroid hormones • Steroid hormones diffuse through plasma membrane and bind to specific receptor in cytoplasm • Activated receptor carries out transduction (response) by becoming a transcription factor or gene activator

46. Regulatory RNAs • Several types of RNA can downregulate gene expression by being complementary to a part of an mRNA or a gene's DNA. MicroRNAs (miRNA) are found in eukaryotes and act through RNA interference (RNAi), where an effector complex of miRNA and enzymes can cleave complementary mRNA, block the mRNA from being translated, or accelerate its degradation. • While small interfering RNAs (siRNA)act through RNA interference in a fashion similar to miRNAs. Some miRNAs and siRNAs can cause genes they target to be methylated, thereby decreasing or increasing transcription of those genes.

47. Basic types of Intergenic sequences • Repetitive elements – when a sequence of two or more nucleotides is repeated many times along the length of a chromosome or chromosomes • Are common, making up nearly half of the human (~44%) • Centromeres, telomeres are made up of repetitive elements • Tandem repeat – repeated sequences are next to each other on the chromosome (10-15% of genome) • Interspersed repeat – repetitions placed intermittently along a chromosome (25-40% of genome) • A repetitive element called the Alu sequence is interspersed every 5,000 base pairs and comprises nearly 5-6 % of total human DNA

48. Basic types of Intergenic sequences • Transposons – DNA sequences that have ability to move within and between chromosomes (10% of genome) • Unique Noncoding DNA (~ half of genome) • Function is unknown • Scientists observed that 74 - 93% of the human genome is transcribed into RNA, so must play some active role in the cell (mRNA, rRNA, tRNA are all transcribed from DNA)

49. New Definition of Gene • A gene is a genomic sequence (DNA or RNA) directly encoding functional products, either RNA or protein

50. Effect of Mutations onProtein Activity • Point Mutations • Involve change in a single DNA nucleotide (substitution) • Changes one codon to a different codon • Affects on protein vary: • Nonfunctional • Reduced functionality • Unaffected • Normal : THE CAT ATE THE RAT • After substitution: THE CAT ATE THE MAT • Frameshift Mutations • One or two nucleotides are either inserted or deleted from DNA • Protein always rendered nonfunctional • Normal : THE CAT ATE THE RAT • After deletion: THE _ATA TET HER AT • After insertion: THE CCA TAT ETH ERA T