Unformatted text preview: 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Central Dogma of Molecular Biology
DNA->RNADNA->RNA->protein Regulation of Genes
Eileen Haase Fall 2007 Lecture 8 All cells, from bacteria to human express genetic human, information this way How do we control which genes are expressed in a cell?
1 2 Many differences between eucaryotic and procarytotic transcription and translation Only procaryotes use a sigma factor that helps RNA polymerase bind DNA. Eucaryotes use a variety of transcription initiation factors and regulatory proteins. 3 4 Control of Genetic Expression Every cell has same DNA
Yet huge differences in gene expression between cell types Cell differentiation arises because cells make and accumulate different RNA and proteins DNA in specialized cell types still contains entire set of genes 5 6 1 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Nucleus of an adult frog skin cell still contains all the DNA needed to control the formation of a tadpole
7 Nature: August 28, 2008 In this immunofluorescent image of an adult mouse pancreas, exocrine cells into which three transcription factors have been inserted are displayed in green. The red areas in the image are insulin. The blue streaks are blood vessels, which are remodeled by and lie close to the new, insulin-producing beta cells. Researchers used poison to destroy beta cells in mice, which made them develop diabetes. The mice were then injected with viruses that slipped into enzyme-producing (exocrine) cells. enzyme Viruses contained transcription factors How does the cell control which genes it expresses?
Control of Genetic Expression 1.Transcription of DNA into RNA 2. RNA processing 3. Translation 4. Protein Activity
10 Within three days of the injection, new insulininsuling g pp secreting cells began to appear. One week later, more than a fifth of the virally infected cells started making insulin. Control starts at initiation site
Promoters of both eukaryotic and prokaryotic cells include the initiation site (AUG start codon) Promoter starts at initiation site and extends ~ 50 nucleotides "upstream" Initiation sites required for RNA polymerase to bind to promoter Regulatory DNA sequences
In addition to promoter, nearly all genes (eukaryotic and prokaryotic) have regulatory DNA sequences that turn gene ON or OFF Can be short (<10 nucleotides in bacteria), or quite long (> 10,000 nucleotides) Gene expression depends upon type of cell, environment, age, and extracellular signals
11 12 2 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Gene regulatory proteins
Proteins that bind to the regulatory DNA sequence (often more than one) Combination of gene regulatory proteins and the regulatory DNA sequence that g y q acts as a switch to control transcription Structure = function: surface of protein fits tightly into double helix of DNA 10% of all genes code for gene regulatory proteins
13 Regulatory Protein Binding -often in major groove of DNA
Gene regulatory p protein attaches to DNA via Hbonds and other weak bonds. Can activate or repress transcription
14 DNA binding motifs in proteins
three motifs are found in gene regulatory proteins in virtually all eukaryotic cells Each motif makes many contacts with DNA Are responsible for controlling the expression of thousands of genes
15 Gene regulatory proteins can be Activators or Repressors
Bind to the operator of a gene (~ 15 nucleotides within the promoter) Repressors turn genes OFF, and activators turn genes ON i Promoter can turn on more than one gene in procaryotic cells These 16 Promoter can alter efficiencies of gene expression `strength' of promoter Transcriptional regulation can be positive or negative
Binding of an activator molecule to the promoter site can increase transcription (ON) Bi di of a repressor molecule t th l l to the Binding f promoter site can decrease transcription (OFF) Releasing an activator from the promoter site can decrease transcription (OFF) Releasing a repressor molecule from the promoter site can increase transcription (ON) Activator binds to regulatory sequence on DNA and interacts with RNA polymerase to initiate transcription 17 18 3 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Modes of Transcriptional Regulation, +/+/- Operon
A cluster of genes arranged together on a single mRNA, along with their promoter and operator. Operator is within the promoter O Operons are common i b t i b t not in bacteria, but t found in eukaryotes, where genes are individually regulated Binding of repressor protein inhibits transcription Binding of activator protein facilitates transcription Binding of repressor molecule inhibits Binding of activator molecule facilitates
19 20 Activator Proteins
Bind to operator and allow initiation of transcription turn a gene ON Repressor Proteins Switches a gene OFF by binding to DNA
No protein made
21 22 Cluster of bacterial genes from a single promoter Promoter is before the start codon
(prior to the coding region for the mRNA) All five genes are transcribed together as a single RNA molecule. The combination of the cluster of genes is an operon
23 Start codon "AUG"
24 4 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Vocabulary
Operon only in prokaryotes a cluster of genes colocalized with a single operator Operator a segment of DNA (about 15 nucleotides) within the promoter. When a ) p promoter. gene regulatory protein binds to this sequence it blocks access of RNA polymerase to the promoter Activator/Repressor protein that binds to another regulatory sequence on DNA
25 Control of genetic expression is different for Eukaryotes vs Procaryotes For prokaryotes, can transcribe an operon and get many different genes at once. Eukaryotes, each gene is individually controlled
26 Differences between prokaryotic and eukaryotic transcription
Eucaryotic RNA polymerases require transcription factors (assemble at the promoter before transcription begins) begins) Repressors and Activators can be quite far away in eukaryotic cells (10,000 nucleotides!) nucleotides!) TATA binding protein
The DNA bending caused by the TATA binding protein may serve as a landmark that helps attract other general transcription factors. TATA box
27 28 Initiation of eukaryotic transcription complex!
Promoter has a "TATA" box 25 nucleotides away from start site (light green) RNA polymerase requires a number of p y q transcription factors (TFIID, TFIIB, etc) RNA polymerase and transcription factors assemble at promoter ATP used to release RNA polymerase from complex and allow it to start transcribing at the start site (in red) Eukaryotic cells cannot initiate transcription without factors
Transcription factors assemble on the promoters Position RNA polymerase correctly p y y Aid in pulling apart two strands of DNA Release RNA polymerase from promoter when transcription begins TFIID (initiation factor) binds to TATA sequence and distorts the DNA
29 5 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Eucaryotic Gene regulatory proteins can control transcription from a distance
Eucaryotic gene activators bound thousands of nucleotides away can increase or decrease activity of RNA polymerase y p y DNA between activator and promoter "loops" out to allow activator proteins and enhancer to come into contact with RNA polymerase Gene regulatory protein bound at distant enhancer stimulates the assembly process for transcription
Can be tens of thousands of base pairs 31 32 Most eukaryotic regulatory proteins work as a "committee"
Need a group of regulatory proteins to bind Regulatory proteins may be located far away p from transcription site Even though control of gene expression is combinatorial, the effect of a single protein can still be decisive. Transcription Initiation Factors and RNA polymerase assemble at the TATA box of the promoter 33 Where everything meets 34 Comparison of Gene activation in bacteria and eukaryotes
Genes 1, 2, and 3 are all transcribed at low levels when activated by their respective gene activator proteins.
Bacteria might have one activator protein turn on many different genes. Eukaryotes might use many different transcription factors and proteins to turn on a single gene 35 36 6 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Cortisol is a steroid hormone that works by activating a gene regulatory protein. Cortisol binds to a receptor protein that undergoes a conformational change which allows it to bind to a specific sequence of DNA activates transcription.
37 Cortisol can activate three different genes when it binds to the glucocorticoid receptor (a gene regulatory protein). The activated gene regulatory protein results in a higher level of transcription of all three genes. 38 Promoter can alter efficiencies of gene expression `strength' of promoter Transcriptional regulation
Many proteins in bacteria are inducible only made when required by the cell for nutritional needs Lac operon Tryptophan There are a large number of proteins involved in the control of gene expression 39 40 No Lactose gene is off lac Operon (Bacterial transcriptional control)
Gene transcription can be switched on and off by gene regulation proteins E. coli can metabolize either glucose or lactose, but prefers glucose Glucose and lactose levels control the initiation of transcription of the lac operon lac operon has genes for enzymes that digest lactose
41 42 LactoseLactose gene is on 7 580.221- Lecture 8 Control of Genetic Expression Fall 2008 lac Operon an "ON" or "OFF" switch In the absence of lactose, a repressor protein binds to the operator, preventing RNA polymerase from transcribing the lac operon's genes. The operon is OFF. When the inducer, lactose, is added, it binds to the repressor and changes the repressor's shape so as to eliminate binding to the operator. As long as the operator remains free of the repressor, RNA polymerase that recognizes the promoter can transcribe the operon's structural genes into mRNA. The operon is ON.
43 lac operon enzymes
Lactose permease spans the cell membrane and uses the energy from the electrochemical gradient to pump lactose into the cell -galactosidase splits lactose into two sugars: glucose and galactose thiogalactoside transacetylase - ?? 44 Lactose Metabolism in Bacteria
Lactose Glucose + Galactose The lac Th l operon t turns on genes to make enzymes for getting lactose into the cell and splitting it lac Operon Model
Transcriptional regulation 3 Genes turned on when lactose is present Molecules structurally similar to lactose can also induce gene (IPTG)
45 46 Lac operon encodes for 3 enzymes that help digest lactose I-gene encodes for the lac repressor protein No lactose present, so repressor protein binds to the lac operon operator and blocks RNA polymerase 47 48 8 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Lac operon is turned off by the repressor protein and no enzymes are made Lactose binds to the repressor protein, so it can no longer bind to the lac operator site (conformational change in the repressor shape) 49 50 Operator is no longer repressed, so RNA polymerase can start transcription of the 3 proteins. 51 52 Mutant lac operon
Lac repressor does not bind to the operator site, even when lactose is present RNA polymerase is always able to initiate p transcription Lactose enzymes are continually transcribed, even transcribed, when there is no lactose present (constitutive) (constitutive) Can introduce wild type repressor on another gene to block operator when lactose is absent (since X Mutant lac repressor cannot bind to the operator on the DNA molecule 53 54 9 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Plasmid DNA codes for functional repressor protein Constitutive always produces proteins, even when lactose is present 55 56 When lactose is present, plasmid repressor binds lactose and frees operator for transcription
Mutant repressor Plasmid repressor Mutant repressor Mutant repressor and Functional Plasmid repressor
57 58 Show Flash Animations
Normal lactose operon regulation Regulation when lac repressor is defective Different kinds of mutations can cause constitutive mutant
Mutation in the repressor protein is a constitutive mutant because it results in continual synthesis of the proteins Mutations in the binding site for the lac repressor (the operator), would prevent the repressor from binding, also resulting in constitutive synthesis 59 60 10 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Constitutive Operator (Oc) "cis-acting" (DNA mediates regulation)
=No inducer CisCis-acting mutations same DNA
Only affect expression of genes on the same DNA molecule in which the mutation occurs Mutations in the promoter, which binds to promoter, RNA polymerase, or the operator, are cisoperator, cisacting CisCis-acting mutations affect binding sites Can't fix with a plasmid
62 ON Operator cannot bind to repressor ON OFF Operator p works =Induced ON Inducer (lactose) binds to repressor 61 transcription lacI (repressor) "trans-acting"
(typically protein mediates regulation)
=No inducer Trans acting mutations - diffuse
TransTrans-acting mutations affect expression of their regulated genes no matter on which DNA molecule in the cell they are located Repressors and Activators are trans-acting transTransTrans-acting proteins can diffuse through the cell and bind to appropriate binding site Can fix with a plasmid with the gene
64 ON lacI- mutant: Operon has no response to IPTG/lactose always ON (=constitutively ON)
ON=protein synthesis =No inducer Both operons OFF= no protein synthesis Plasmid restores operon's response to IPTG & lactose
63 Tryptophan repressor is an allosteric protein
When tryptophan binds to repressor, get 3D change in shape so repressor can bind to operator DNA and stop transcription Tryptophan repressor protein is always present in the cell (constitutive) (constitutive) Conformational change of Trp repressor by binding
tryptophan 65 A dimer (two molecules of tryptophan bound to the two molecules of the repressor) binds to the DNA and blocks transcription of the gene that makes tryptophan 66 11 580.221- Lecture 8 Control of Genetic Expression Fall 2008 [Tryptophan] can turn genes off [Tryptophan] controls its own gene expression
Tryptophan gene encodes proteins required to make tryptophan if it is not in the diet Repressor protein is inactive at low levels of tryptophan, gene is t t t h i turned ON d High levels of tryptophan bind to repressor protein and activate it. Turn gene OFF When [tryptophan] , it is released from the [tryptophan] repressor protein and the gene is turned back on.
68 Repressor binds to tryptophan and blocks transcription of the enzymes needs to make tryptophan. RNA polymerase only works if the concentration of tryptophan 67 is low. cAMP is a "hunger" signal
Indicates cell ATP has been used (ATPADPAMP (ATPADPAMPcAMP) [glucose] is low CAP catabolic activator protein binds to cAMP Max transcription of lac operon requires presence of cAMPcAMP-CAP complex
69 Cell has no lactose and can eat glucose not starving. No need to make lactose enzymes Cell has glucose and lactose to eat prefers glucose. Not starving. Cell is starving, lots of cAMP! No glucose, only lactose availablemake lactose enzymes No lactose, Glucose -No lac mRNA Lactose, Glucose and [cAMP] No glucose, [cAMP] and lactose 70 Cooperative binding of cAMP-CAP & RNA cAMPpolymerase have greater affinity together than either protein alone Maximum expression of lactose enzymes when glucose, lactose, and cAMP glucose lactose cAMP RNA polymerase binds more stably to DNA promoter with cAMP-CAP
71 Get a little transcription with lactose present. Get a lot of transcription with no glucose, lots of lactose, and lots of cAMP 72 12 580.221- Lecture 8 Control of Genetic Expression Fall 2008 How can you tell where everything binds?
Operators Promoters Repressors/Activators Initiation sites Ribosomes DNA Footprinting
Detects proteins that bind to a DNA regulatory element When Wh protein is bound to DNA, it protects t i i b d t DNA t t DNA from digestion by nucleases The appearance of a footprint indicates the presence of a transcription factor that binds to the control element 73 74 Gel Analysis: DNA Footprinting Method
Samples of DNA fragments are labeled at one end Digested in the presence and absence of a DNA-binding p DNAg protein Denatured, electrophoresed, and run through electrophoresed, gel Region protected by bound protein appears as a "gap" or "footprint" since these areas are protected from the DNAse
75 Proteins migrate through a gel at a rate that is proportional to their charge and size 76 DNA footprinting of binding proteins combines 2 methods:
DNA sequencing to determine the precise order of the nucleotides Footprinting to determine where in the sequence the binding protein attaches Footprints of lac control region DNA 77 RNA polymerase bound to DNA 78 13 580.221- Lecture 8 Control of Genetic Expression Fall 2008 Packing of promoter DNA into nucleosomes can affect initiation of transcription
How do the gene regulatory proteins, transcription factors, and RNA polymerase gain access to DNA that is packed into nucleosomes? How does packing affect the initiation of transcription? More compact forms of chromatin are resistant to transcription initiation
79 80 Control of Genetic Expression 1.Transcription of DNA into RNA 2. RNA processing 3. Translation
Remove introns Eukaryotic mRNA: 5' cap and poly A tail of several hundred nucleotides 4. Protein Activity
Ready for cytoplasm!
81 82 Alternative splicing/examples can make different combinations of mRNA from the same gene by using different exons Primary transcript can be spliced in different ways for specific cell types 83 84 14 580.221- Lecture 8 Control of Genetic Expression Fall 2008 mRNA lifetime varies (30 min 10 hrs) The longer mRNA lasts, the more protein made. Possible signal in mRNA near the poly A tail helps determine lifetime.
85 Control of Genetic Expression 1.Transcription of DNA into RNA 2. RNA processing 3. Translation - binding and dissociation of initiation factors on ribosomal subunit 4. Protein Activity
86 Ribosomal unit needs initiation factors to start translation (eukaryotic) Control of Genetic Expression 1.Transcription of DNA into RNA 2. RNA processing 3. Translation 4. Protein Activity Blood clotting Pepsinogen - digestive enzyme that breaks
down proteins Angiotensin helps control blood pressure
87 88 Protein Activity depends upon:
-[protein] = rate made [p ] rate destroyed -Post translational processing (folding pH) Control of Genetic Expression 1.Transcription of DNA into RNA 2. RNA processing 3. Translation 4. Protein Activity QUESTIONS???
89 90 15 ...
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- DNA, rna polymerase