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CONCEPT: CONTROL OF GENE EXPRESSION BASICS Gene expression is the process through which cells selectively to express some genes and not others Every cell in an organism is a clone because they all contain an identical copy of DNA - Different appearances and function depends on selectively expressing certain genes and not others Gene expression controls the expression of proteins and RNAs Cell differentiation is the process by which a cell becomes for a particular function - Differentiation is entirely directed by gene expression control - Allows for the development of multicellular organisms with diverse cell types EXAMPLE: Differentiation of a stem cell into many different blood cell types Gene expression can be at various steps in the DNA to RNA to Protein pathways Gene expression can be controlled by external signals - One example is through hormones Housekeeping genes are genes expressed in every cell because they are critical for cellular life - Examples include: Ribosomal genes, RNA polymerase genes, DNA repair genes Page 2
EXAMPLE: The many ways gene expression can be controlled PRACTICE: 1. Gene expression is defined as which of the following? a. Genes are expressed because each cell contains a different set of genes b. Choosing which genes are expression by regulating only transcription c. Choosing which genes are expression because on only internal signals d. Cells choosing to express some genes and not others at many steps in the DNA to protein pathway Page 3
2. True or False: Every gene is regulated differently in each cell type. a. True b. False Page 4
CONCEPT: EPIGENETIC REGULATION OF GENE EXPRESSION Epigenetic modifications Modifications of proteins can effect DNA structure and control gene expression Histone methylation is the addition of a methyl group to certain amino acids on the histone protein - CpG (CG) islands are CG nucleotides that generally remain unmethylated - Surround promoter regions ~ high CG content ~1000-2000 nucleotides long - Other CG sites in the genome attracts the methyltransferase and histone methylation - gene expression by stimulating chromatin condensation Histone acetylation is the addition of an acetyl group to certain amino acids on the histone proteins - Found in transcriptionally active chromatin because it stimulates an open chromatin structure - Catalyzed by histone acetyltransferase and removed by histone deacetylase (HDAC) The histone code is the combination of methylation and acetylation events that regulate gene expression - Recruit other regulatory proteins to the gene EXAMPLE: CpG islands Certain proteins can act as genetic activators or repressors Gene activator or repressor proteins can modify local chromatin structure to support gene expression Page 5
- Nucleosome (chromatin) remodeling factors are proteins that alter the arrangement of nucleosomes - Do not effect methylation or acetylation - Act by moving the histone protein octamer to a different DNA location - Elongation factors: include histone modifying enzymes that disrupt nucleosomes for transcription - These proteins typically reside on the RNA polymerase, so that they can act during transcription Activator proteins work synergistically - Transcriptional synergy is when several activator proteins increase the rate of transcription - Occurs when the new rate is higher than the rate sums of each activator working alone EXAMPLE: Activated and condensed chromatin Epigenetic Heredity Cells terminally differentiate, meaning that after differentiation, the cell progeny that cell type Cell memory is the property that allows cells to pass patterns of gene expression to their offspring - This is heredity that doesn t include the DNA sequence but instead the chromatin modifications Epigenetic inheritance is the property that allows organisms to pass patterns of gene expression to offspring - This is heredity that doesn t include the DNA sequence but instead the chromatin modifications - Genomic imprinting is when one parental gene copy remains active, while the other remains inactive Page 6
- Inactive copies remain methylated depending on source (sperm or egg) - Two identical DNA sequences, but different chromatin modifications which effect expression EXAMPLE: Altered methylation status of one gene (A vy gene) causes different phenotypes in genetically identical mice Chromosome wide structures can also be inherited by cellular offspring X-inactivation is the transcriptional inactivation of an entire X chromosome X-inactivation initiation is random, meaning that both X copies have the same chance of being inactivated - Once one has been chosen it remains inactive for all cellular division X-inactivation initiation occurs after a few several thousand cells have formed - Therefore a mosaic phenotype appears when these cells each choose different X chromosomes - The genes on each copy encode for a different appearance, which can be seen throughout the body EXAMPLE: Calico cats are the result of X-inactivation Page 7
PRACTICE 1. Which of the following is not a form of epigenetic heredity? a. Cell memory b. X inactivation c. CpG islands d. Genomic imprinting 2. Which of the following modifications is found most often with open chromatin? a. Acetylation b. Methylation c. Phosphorylation d. Ubiquitination Page 8
3. True or False: Chromatin remodeling factors move nucleosomes by altering the methylation or acetylation pattern. a. True b. False 4. CpG islands are groupings of CG nucleotides that are unusual because why? a. They are highly methylated b. They are unmethylated c. The are found in genes d. They are highly acetylated Page 9
CONCEPT: TRANSCRIPTIONAL REGULATORS OF GENE EXPRESSION How Transcriptional Regulators Work Transcriptional regulators gene expression by activating or repressing the transcription of genes Transcriptional repressors turn genes off and therefore inhibit transcription - Can compete with activators for binding - Can inhibit transcription via protein-protein interactions Transcriptional activators turn genes on, and therefore activate transcription - Help make promoters fully functional by connecting with RNA polymerase Can work with coactivators or corepressors which help to control transcription - Modifying chromatin structure - Activating the regulatory protein Mediator is a 24 subunit complex that acts as a connector between regulatory proteins and RNA polymerase EXAMPLE: Comparison of activators and repressors Activator Gene Repressor Gene Rarely do they work alone, and require other interactions and to be fully functional - Other transcription factors are recruited to regulate gene expression - General transcription factors: bind to core promoter site (Ex: TFIIB, TFIIH) - Sequence specific factors bind to regulatory sites to activate/repress expression - Each gene is regulated differently Page 10
EXAMPLE: Combination of transcription factors results in gene regulation DNA binding motifs There are four common DNA binding that transcriptional regulators contain Helix-turn-Helix: One helix makes contact with the DNA, while the other helix stabilizes the interaction - Homeodomains are found on Hox genes which are crucial for proper development Zinc Finger: Has repeats of cysteine and histidine that bind zinc and fold into a finger-like structure to bind DNA Leucine Zipper: Dimerization of alpha helices with many leucine residues can bind DNA Helix-loop-Helix: Two alpha helices connected by a loop can bind DNA Transcriptional regulators bind to regulatory DNA sequences between 10 and 10,000 nucleotides in length - Regulator proteins are degenerate meaning they don t need an exact sequence to bind - They don t necessarily bind to the DNA nucleotides can recognize and noncovalently bind to the helix EXAMPLE: DNA binding domains Helix-Turn-Helix Zinc Finger Leucine Zipper Helix-Loop-Helix Page 11
Prokaryotes use RNA polymerase subunits to control gene transcription Sigma subunit of RNA polymerase is required to recognize a promoter - Many sigma subunits exist and each recognizes a different set of promoters Gene expression is controlled by replacing the sigma subunits of RNA polymerase EXAMPLE: Sigma factor replacement allows for gene regulation in prokaryotic cells RNA polymerase Three different sigma factors Types of Transcriptional Regulators Transcriptional regulators can to sequences located near or far from the gene they re regulating Promoter-proximal elements lie near to the promoter site - Promoter binds RNA polymerase and orients it correctly so it can transcribe the gene - Contains the initiation site where RNA synthesis begins EXAMPLE: A promoter (yellow) recruiting RNA polymerase (green) to the gene (blue) Page 12
There are numerous regulators that bind to DNA sequences from the gene - Enhancer is a DNA site to which gene activators bind - Can be upstream or downstream from gene, and usually 1000s nucleotides away from promoter - DNA between enhancer and promoter loop out to allow the two regions to interact - Silencers is a DNA site to which gene repressors bind. Acts similarly to an enhancer Insulators (barrier elements) divide chromosomes into independent segments - Prevents distant elements (enhancers) from acting on promoters in a different segment Gene control region: entire DNA sequence involved in regulating and initiating transcription of a gene EXAMPLE: Example of enhancer activation Tryptophan Repressor and Lac Operon The amino acid is a major regulator of gene expression in prokaryotes Can bind to operons (stretches of many related genes) and inhibit transcription - Tryptophan binds to a transcriptional repressor to activate it - The activated repressors binds to regulatory sequences to inhibit genes involved in tryptophan creation Page 13
Allows gene expression to be controlled by environmental levels of tryptophan EXAMPLE: Control of the trp operon The lac operon controls the of lactose in E. coli No lactose available: The lac repressor binds and halts transcription of lac operon Glucose available: the activator Catobolie activator protein (CAP) remains inactive, but no direct repression Lactose available: the activator Catobolie activator protein (CAP) binds upstream of promoter and activates EXAMPLE: Control of the lac operon Page 14
PRACTICE 1. Which of the following is not a DNA binding motif? a. Zinc Finger b. Leucine Zipper c. Helix-loop-Helix d. Helix-zipper-Helix 2. What is the purpose of a transcriptional mediator? a. To mediate regulation between transcription and translation b. To mediate the process of transcription c. To mediate between regulatory proteins and RNA polymerase d. To mediate between RNA polymerase and DNA Page 15
3. True or False: Enhancers can reside downstream of the gene they regulate. a. True b. False 4. If lactose is present, what happens to the lac operon? a. It is activated b. It is repressed Page 16
5. Page 17
CONCEPT: ACTION OF TRANSCRIPTION REGULATORS Combinatorial Control Combination of regulator proteins gene expression Multiple proteins work together to determine the expression of a single gene - Usually the first protein has high affinity, and then binding increases the affinity of other proteins - Limits the number of transcription regulators needed Expression can be decided by a single regulator protein - Works like an on/off switch by completing the combination Combinations can control the generation of different cell types - A few transcription regulators control sets of genes resulting in cell differentiation Combinations can be controlled by environmental signals - Response elements are DNA sequence in a promoter that can bind to regulator proteins - Ex: Heat-shock response elements, hormones EXAMPLE: Combinations of regulatory proteins control gene expression Promoter Promoter RNA pol. Gene Steps to Gene Activation Gene activation occurs in 6 steps 1. Regulatory proteins bind to an enhancer 2. This binding stimulate the DNA to form a loop which connects the enhancer and promoter 3. Activators interact with coactivators to alter chromatin structure 4. Activators interact with the mediator Page 18
5. Mediator facilitates the correct positions of RNA polymerase 6. RNA polymerase begins transcribing EXAMPLE: Steps to gene activation 1. Enhancer Promoter Gene Enhancer Promoter Gene 2. Promoter Gene 3 5 6. 4 RNA pol. Gene Promoter Nuclear Receptors and Hormones Nuclear receptors are transcriptional regulators that sense hormone (steroids) and regulate gene expression Nuclear receptors contain a few important - N-terminal act as an activation domain - Has a DNA binding domain Inverted repeats are sequences of nucleotides followed by a reverse complement downstream - Hormone response elements are inverted repeats that many nuclear receptors bind Page 19
EXAMPLE: Hormone activation of a gene PRACTICE 1. Choose all of the following factors involved in combinatorial control of gene expression. a. Regulatory proteins b. Response elements c. Histone Proteins d. RNA polymerase e. Glycosylation Page 20
2. What are nuclear receptors? a. Hormones b. Receptors found on the surface of the nucleus c. Receptors found on the surface of the cell d. Proteins in the nucleus that bind to hormones Page 21
CONCEPT: POST TRANSCRIPTIONAL REGULATION RNA Processing, Translation, and Degradation Regulation of mrna after transcription is a major way to gene expression RNA processing includes alternative splicing, preparation for nuclear export, and RNA editing - Improperly processed mrnas are not exported and translated RNA translation can be controlled - Phosphorylation of eifs (bind to 3 mrna to promote translation) can globally inhibit cellular translation - Phosphorylated eif cannot exchange GDP for GTP and therefore can t promote translation - Translational repressors are proteins that control translation of specific mrnas mrna degradation vary and are one way to regulate gene expression - Shorter poly(a) tails are less stable than longer tails - Exosomes degrade mrna from 3 to 5 via exonucleases - P bodies are nuclear mrna processing bodies that degrade mrna from 5 to 3 - Nonsense mediated decay degrades improperly spliced mrna that lack proper protein coding regions - When stop codon is in wrong place EXAMPLE: RNA processing, translation, and degradation can control gene expression Page 22
RNA Interference Various types of regulatory RNAs can control gene expression Small interfering RNA (sirna) is one form of RNA mediated inhibition evolved to cells from viruses 1. sirnas are double stranded RNA that enter cells via foreign objects 2. The enzyme dicer cleaves sirnas into small fragments 3. The RNA induced silencing complex (RISC) binds these fragments and degrades one strand 4. The single stranded sirna can bind to a complementary mrna which is then degraded by RISC - Argonaute is the catalytic component of RISC that cleaves the mrna EXAMPLE: sirna mediated mrna degredation Page 23
Micro RNA (mirna) is a second form of RNA mediated inhibition that is by the genome 1. mirnas are single stranded RNAs created through transcription (~22 nucleotides long) 2. After transcription, mirnas form hairpins or loops based on complementary RNA sequences 3. DROSHA cleaves the loops and the free mirna associates with RISC 4. The processed mirna binds to a 3 UTR end of mrna and inhibits expression via RISC degradation - Each mirna can regulated ~200 mrnas EXAMPLE: mirna processing and complex formation with RISC Page 24
Other noncoding RNAs regulate gene expression - Piwi-interacting RNA (pirnas) suppress movement of transposons - Long noncoding RNAs are 200+ nucleotides in length and regulate gene expression Protein Regulation Regulation of mrna after transcription is a major method of controlling gene expression Protein modifications can inhibit or activate protein - Examples include: Protein phosphorylation, dephosphorylation and cleavage Protein degradation controls a proteins function - Ubiquitin labeling - proteasome destruction and lysosomal destruction - Degrons are protein regions that control a protein s destruction EXAMPLE: Presence of degron reduces protein presence over time Page 25
PRACTICE 1. Choose all of the following post-transcriptional regulators of gene expression. a. Micro RNAs b. sirnas c. Cleavage d. Exosomes 2. True or False: When the sirna interacts with RISC for the first time it is single stranded. a. True b. False Page 26
3. What is the name of the enzyme that cleaves the mirna in the nucleus before it travels to the cytoplasm to exert its effects? a. RISC b. Argonaut c. DROSHA d. RNA Polymerase 4. What is the name of the region on a protein that controls its degradation over time? a. Degradation sequences b. Ubiquitin c. Ubiquitin binding site d. Degron Page 27
5. True or False: All non-coding RNAs are responsible for regulating gene expression. a. True b. False Page 28