Control of Gene Expression 1
How Gene Regulation Works 2
Control of Gene Expression Controlling gene expression is often accomplished by controlling transcription initiation Regulatory proteins bind to DNA May block or stimulate transcription Prokaryotic organisms regulate gene expression in response to their environment Eukaryotic cells regulate gene expression to maintain homeostasis in the organism 3
Regulatory Proteins Gene expression is often controlled by regulatory proteins binding to specific DNA sequences Regulatory proteins gain access to the bases of DNA at the major groove Regulatory proteins possess DNA-binding motifs 4
Prokaryotic regulation Control of transcription initiation Positive control increases frequency of initiation of transcription Activators enhance binding of RNA polymerase to promoter Negative control decreases frequency Repressors bind to operators in DNA 5
Prokaryotic cells often respond to their environment by changes in gene expression Genes involved in the same metabolic pathway are organized in operons Induction enzymes for a certain pathway are produced in response to a substrate Repression capable of making an enzyme but does not 6
lac operon Contains genes for the use of lactose as an energy source -b-galactosidase (lacz), permease (lacy), and transacetylase (laca) Gene for the lac repressor (laci) is linked to the rest of the lac operon 7
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP-binding site Promoter for I gene PI Gene for repressor protein I CAP P lac Regulatory region Promoter for lac operon O Operator Genefor ß-galactosidase Z Gene for permease Y Gene for transacetylase A lac Control system Coding region 8
The lac operon is negatively regulated by a repressor protein lac repressor binds to the operator to block transcription In the presence of lactose, an inducer molecule (allolactose) binds to the repressor protein Repressor can no longer bind to operator Transcription proceeds Even in the absence of lactose, the lac operon is expressed at a very low level 9
Glucose repression Preferential use of glucose in the presence of other sugars Mechanism involves activator protein that stimulates transcription Catabolic activator protein (CAP) is an allosteric protein with camp as effector Level of camp in cells is reduced in the presence of glucose so that no stimulation of transcription from CAP-responsive operons takes place Inducer exclusion presence of glucose inhibits the transport of lactose into the cell 10
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Glucose Low, Inducer Present, Promoter Activated DNA Allolactose CAP camp camp CAP binds to DNA Glucose level is low camp is high CAPbinding site camp Repressor will not bind to DNA mrna a. camp activates CAP by causing a conformation change RNA polymerase is not blocked and transcription can occur 11
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Glucose High, Inducer Absent, Promoter Not Activated Glucose is available camp level is low CAP does not bind Repressor binds to DNA Effector site is empty, and there is no conformation change RNA polymerase is blocked by the lac repressor b. 12
trp operon Genes for the biosynthesis of tryptophan The operon is not expressed when the cell contains sufficient amounts of tryptophan The operon is expressed when levels of tryptophan are low trp repressor is a helix-turn-helix protein that binds to the operator site located adjacent to the trp promoter 13
The trp operon is regulated by the trp repressor protein trp repressor binds to the operator to block transcription Binding of repressor to the operator requires a corepressor which is tryptophan Low levels of tryptophan prevent the repressor from binding to the operator 14
Tryptophan repressor Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Tryptophan 3.4 nm 15
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Tryptophan Absent, Operon Derepressed trp repressor cannot bind to DNA Inactive trp repressor (No tryptophan present) Operator mrna Translation E D C B A Enzymes for tryptophan synthesis produced a. Gene for trp repressor Promoter for trp operon RNA polymerase is not blocked, and transcription can occur 16
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Tryptophan Present, Operon Repressed Tryptophan Tryptophan binds to repressor, causing a conformation change Repressor conformation change allows it to bind to the operator RNA polymerase is blocked by the trp repressor, and transcription cannot occur Enzymes for tryptophan synthesis not produced Gene for trp repressor b. 17
Eukaryotic Regulation Control of transcription more complex Major differences from prokaryotes Eukaryotes have DNA organized into chromatin Complicates protein-dna interaction Eukaryotic transcription occurs in nucleus Amount of DNA involved in regulating eukaryotic genes much larger 18
Transcription factors General transcription factors Necessary for the assembly of a transcription apparatus and recruitment of RNA polymerase II to a promoter TFIID recognizes TATA box sequences Specific transcription factors Increase the level of transcription in certain cell types or in response to signals 19
Interactions of various factors Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA Polymerase II B TAFs F TFIID E Transcribed region H TATA box A Core promoter 20
Promoters form the binding sites for general transcription factors Mediate the binding of RNA polymerase II to the promoter Enhancers are the binding site of the specific transcription factors DNA bends to form loop to position enhancer closer to promoter 21
Enhancers Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Activator Transcription factor RNA polymerase Transcribed region Promoter Enhancer TATA box mrna synthesis 22
Transcription complex Few general principles Nearly every eukaryotic gene represents a unique case Great flexibility to respond to many signals Virtually all genes that are transcribed by RNA polymerase II need the same suite of general factors to assemble an initiation complex 23
Transcription complex Enhancers Coding region Activator Enhancer Coactivator Activator B General factors TAFs TFIID F E RNA polymerase II Activators These regulatory proteins bind to DNA at distant sites known as enhancers. When DNA folds so that the enhancer is brought into proximity with the initiation complex, the activator proteins interact with the complex to increase the rate of transcription. Coactivators H These transcription factors stabilize the transcription complex by bridging activator proteins with the complex. A General Factors These transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to initiate transcription. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24
Eukaryotic chromatin structure Structure is directly related to the control of gene expression DNA wound around histone proteins to form nucleosomes Nucleosomes may block access to promoter Histones can be modified to result in greater condensation 25
Methylation once thought to play a major role in gene regulation Many inactive mammalian genes are methylated Lesser role in blocking accidental transcription of genes turned off Histones can be modified Correlated with active versus inactive regions of chromatin Can be methylated found in inactive regions Can be acetylated found in active regions 26
Condensed solenoid Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nucleosomes can block the binding of RNA polymerase II to the promoter Amino acid histone tail N-terminus Addition of acetyl groups to histone tails remodel the solenoid so that DNA is accessible for transcription Acetyl group DNA available for transcription 27
Chromatin-remodeling complexes Large complex of proteins Modify histones and DNA Also change chromatin structure ATP-dependent chromatin remodeling factors Function as molecular motors Catalyze 4 different changes in DNA/histone binding Make DNA more accessible to regulatory proteins 28
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ATP ADP+ P i ATP-dependent remodeling factor 1. Nucleosome sliding 2. Remodeled nucleosome 3. Nucleosome displacement 4. Histone replacement 29
Posttranscriptional Regulation Control of gene expression usually involves the control of transcription initiation Gene expression can be controlled after transcription with Small RNAs mirna and sirna Alternative splicing RNA editing mrna degradation 30
Protein Degradation Proteins are produced and degraded continually in the cell Lysosomes house proteases for nonspecific protein digestion Proteins marked specifically for destruction with ubiquitin Degradation of proteins marked with ubiquitin occurs at the proteasome 31
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ubiquitin Protein to be destroyed ATP ADP + P i Destroyed by proteolysis 32
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Degradation Polypeptide fragments Ubiquitination Proteasome ATP ADP + P i Ubiquitin ADP + P i ATP Targeted protein ADP ATP Ubiquitin ligase 33