Chapter 18: Regulation of Gene Expression

1. Chapter 18: Regulation of Gene Expression What we will cover: 1. How Proks regulate genes based on environment 2. How Euks regulate gene expression to maintain different cell types 3. How gene regulation can allow a single cell (zygote) to become a fully functioning organism of many cell types.

2. General Information: • Gene Expression – from genotype to phenotype (gene to protein) • Genes are turned “on” and “off” (transcribed or not transcribed) • First understanding of gene control came from E. coli which changes activity based on environmental changes.

3. Gene Regulation in Prokaryotes OPERONS

4. I. Gene Regulation in Prokaryotes (first seen in E.coli) A. E.coli lives in an erratic environment in your intestines. 1. if deprived of amino acid tryptophan, it responds by activating a metabolic pathway to make its own tryptophan 2. If you eat a tryptophan-rich meal (Thanksgiving) it stops producing tryptophan thus saving resources. 3. Metabolic control occurs at two levels: a. vary the # of specific enzymes made b. adjust the activity of enzymes already present B. Operons = 1. Only exist in prokaryotes 2. Expression of genes can be coordinated 3. Enable prokaryotes to thrive in changing environment

5. C. The basic parts of an operon (always on the AP Exam) 1. Promoter – where RNA polymerase attaches and initiates transcription 2. Operator – segment of DNA positioned within the promoter or between the promoter and coding genes. Acts as a “switch” by determining whether RNA polymerase can attach to the promoter or not. 3. Genes of the operon – have related functions (ex: all will be involved in the synthesis of tryptophan)…benefit is they are easily coordinated 4. Repressor – protein that binds to the operator blocking the attachment of RNA polymerase to the promoter (no transcription) the gene is “off.” 5. Regulatory gene – located outside the operon, codes for the repressor…it is expressed continually

7. D. Three kinds of Operons (more details to follow) 1. Inducible Operon -- “repressor active alone” a. inducer inactivates repressor b. lac operon 2. Repressible Operon -- “repressor inactive alone” a. corepressor activates repressor b. trp operon 3. Activators (type of positive gene regulation) a. It is not enough for lactose to be present for bacteria to break it down…glucose must also be in short supply. How does it know? b. cyclic AMP (cAMP) accumulates when glucose is absent. c. when cAMP bind allosterically to cAMP regulatory protein (CAP) it becomes active and binds next to the lac promoter. This makes it easier for RNA polymerase to bond. d. lac operon is under dual control (negative control by the lac repressor and positive control by the CAP) Negative gene regulation

9. E. Inducible Operon (example: the lac operon in E. coli) 1. E. coli can use lactose to make energy. This operon codes for 3 enzymes that metabolize lactose. 2. Operator = a. determines whether or not RNA polymerase can attach b. When operon is turned “off” lactose is not present so a repressor molecule binds to the operator. c. When operon is turned “on” lactose is present in the cell’s environment causing allolactoase (an isomer of lactose) to bond to the allosteric site of the repressor. Therefore causing the repressor to release from the operator. Allolactose is an “inducer” – inactivates the repressor 3. Enzymes made from this type of regulation are called “inducible enzymes”, because their synthesis is induced by a chemical signal.

11. Arabinose Operon • Works exactly the same way as the Lactose Operon. • Instead of digestive enzymes you make GFP (green florescent protein) • This is the operon used in the Transformation lab (pGLO lab)

12. Lac Operon

13. F. Repressible Operon – (example: the trp operon in E.coli) 1. E.coli makes tryptophan (when it is not in the environment) in a series of steps, each catalyzed by a specific enzyme 2. The trp repressor is made in an inactive form with little affinity for the trp operator. a. if tryptophan binds to the repressor at the allosteric site, the repressor is activated and can attach to the operator. b. Tryptophan functions as a corepressor – a small molecule that cooperates with a repressor protein to switch an operon off. 3. If tryptophan levels drop, repressor is released and transcription of the operon’s genes resumes. 4. Example of feedback inhibition…keeps the substrate at a constant level.

15. ?A certain mutation in E. coli impairs the ability of the lac operator to bind to the repressor. How would this affect the cell?

16. Gene Expression in Eukaryotes Chromatin Modification Transcription RNA processing Translation mRNA degradation Protein processing and degradation Noncoding RNA’s

17. II. Chromatin structure is based on levels of DNA packing (pg.320…chapter 16) A. Nucleosomes 1."Beads on a String”. 2. DNA wound on a protein core. 3. Packaging for DNA. 4. Controls transcription. B. 30-nm Chromatin Fibers 1. A cylinder of tightly coiled nucleosomes 30 - nm in diameter. C. Looped Domains 1. Loops of 30 - nm chromatin D. Chromosomes 1. Large units of DNA. 2. Heterochromatin – region of highly condensed chromatin; areas that are not transcribed. 3. Euchromatin – region of less condensed chromatin; areas of active transcription. Protein Scaffold

19. III. Gene Expression in Euks Gene expression can occur at any step from gene to protein  Potential control points where gene expression can be turned on or off, sped up or slowed down. a. chromatin changes that unpack the DNA b. transcription (most common) c. RNA processing d. translation e. altering protein product  Differential Gene Expression -- A typical human cell expresses about 20% of its genes at any given time (some fewer than that)

20. A. Regulation of Chromatin Structure 1. Packing of DNA prevents gene expression a. RNA polymerase can not bond when DNA is packed (heterochrome region) b. Example: “X chromosome inactivation”  One of the two X chromosomes in each cell is inactivated at random  A female that is heterozygous for genes on the X chromosome, has cells that express different alleles. 2. Histone Modifications (Histone Acetylation/deacetylation) a. attachment or removal of acetyl group (-COCH3) to or from certain amino acids of histone proteins b. acetylated histone of a nucleosome changes shape so their grip on DNA is less tight. (transcription easier) c. enzymes that do this are closely associated with transcription factors (may be coupled reactions)

21. 3. DNA methylation – attachment of methyl groups (-CH3) to DNA bases (usually cytosine) after DNA is made. a. genes not expressed – heavily methylated (removing them in lab turns the genes on) b. methylating enzymes act at DNA sites where one strand is already methylated, thus correctly methylating the daughter strand. c. Accounts for Genomic Imprinting in mammals – permanently regulating expression of either the maternal or paternal allele of certain genes at the start of development. 4. Epigenetic Inheritance – inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence a. modifications to chromatin can be reversed.

22. B. Regulating Transcription Initiation 1. Regulatory proteins bind to DNA to turn transcription of genes on or off 2. In contrast to operons, each Euk gene has its own promoter and other control sequences. 3. The “default” state of most genes is off a. exception, housekeeping genes… b. therefore, activators are more important than repressors. c. Activators = transcription factors that bind to “enhancer region” of DNA d. Enhancer Region = DNA sequence that recognizes certain transcription factors that can stimulate transcription of genes (usually 1000’s of nucleotides away)

24. 4. Turning on a Euk gene involves regulatory proteins called transcription factors plus RNA polymerase. a. Activators bind to enhancer region of DNA b. Enhancers are far away from gene it regulates c. DNA then bends forming a complex with other transcription factors and the gene’s promoter region. d. Silencers = 5. Coordinating Euk gene expression a. depends on association of specific enhancer or collection of enhancers

25. Only liver cells make Albumin. • Only Lens cells make Crystallin. • Both cells contain genes to do both. • The specific transciption factors made in the cells will determine which genes are expressed. • How do sets of Activators come to be present in different cells?(answered on slide 29)

26. C. Post Transcriptional Regulation 1. RNA Processing -- “alternative splicing” 2. mRNA degradation a. life span of mRNA varies b. begins with shortening of poly A tail which triggers enzymes to remove 5’ cap (regulated by nucleotide sequences in the mRNA in “leader” portion of mRNA) c. once cap is removed, nuclease enzymes rapidly breakdown mRNA d. miRNAs (microRNAs) – made from longer pieces of RNA that folds on itself to make a double-stranded hairpin structure. Enzyme called “dicer” cuts the strand into short pieces. One of the two strands is broken down while the other associates with a large protein to allow it to bind to mRNA which degrades mRNA or blocks translation (this is called RNA interference (RNAi) and is caused by “small interfering RNAs (siRNAs)

27. 3. Translation initiation a. most control mechanisms block initiation b. proteins with regulatory functions bind to specific sequences within the leader region preventing ribosome attachment c. control here can lead to “global” control (skip to picture) Global Control in a Red Blood Cell Global control is also important in embryonic development. (eggs have mRNA that are not translated until after fertilization) Global control in plants: store mRNA in darkness…light triggers reactivation of translation

28. D. Post-translation control (protein processing and degradation) 1. cutting Euk polypeptides into smaller, active final products (example: insulin) (never cut) 2. some proteins require additions (can be blocked) 3. some proteins need to go to specific location of cell to function…(never get there…example: cystic fibrosis) 4. Selective breakdown of proteins a. to mark protein for destruction, cell attaches a small protein called ubiquitin to the protein b. Proteasomes = giant protein complexes that recognize ubiquitin and degrade the tagged protein

29. IV. Noncoding RNAs (aka non-protein-coding RNAs) 2006 Nobel Prize A. mRNA translation 1. microRNAs (miRNAs) bind to complementary sequences in mRNA and degrades it or blocks translation. 2. estimated that 1/3 of our genes are regulated this way. 3. RNA interference (RNAi) – using synthetic double-stranded RNA molecules that match the sequence of a particular gene to trigger the breakdown of the gene’s mRNA a. small interfering RNAs (siRNAs) 4. miRNA formed from a single hairpin in a precursor RNA siRNA formed from longer double-stranded RNA molecules 5. Induction B. Chromatin configuration 1. siRNAs play a role in forming heterochromatin

31. V. Genetic Control of Embryonic Development A. Three processes: 1. Cell division 2. Cell differentiation 3. Morphogenesis B. Revisit question from slide 22 (how do different sets of activators come to be present in different cells) 1. Materials in the egg set up a sequential program of gene regulation that occurs as cells divide…makes the cells become different from each other in a coordinated fashion. 2. Two sources of information tell a cell which genes to express at any given time during embryonic development, which leads the cell to differentiate a. egg’s cytoplasm b. the environment around the cell

32. 3. The egg’s cytoplasm a. Contains RNA and proteins from the mother’s DNA b. Things in the egg are distributed unevenly in the cytoplasm -- “cytoplasmic determinants” -- c. After the zygote divides, the combination of cytoplasmic determinants in a cell helps determine the cell’s fate. 4. The environment around the cell a. more important as the number of cells increases b. Induction occurs –

33. 5. Determination –the events that lead to the observable differentiation of a cell. a. once a cell goes through determination, the embryonic cell is irreversibly committed to its final fate. b. This is “marked” by the expression of genes for tissue specific proteins c. When the cell starts making mRNA’s for those proteins, it is considered “differentiated”

34. C. Setting up the Body Plan (head vs. tail) 1. Pattern formation— a. head, tail, back, front, right and left have to be determined before organs can form b. “positional information” is provided by cytoplasmic determinants and inductive signals c. cues tell cell its location relative to the “axes” which will in turn, determine how it will respond to future signals 2. The fruit fly was extensively studied

35. Homeotic genes Segmentation genes Maternal effect gene (aka “egg-polarity genes”) EX: Bicoid gene Morphogens