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Control of Gene Expression Chapter 16

3 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

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

5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Vantage point = Hydrogen bond donors = Hydrogen bond acceptors = Hydrogen methyl group = Hydrogen atoms unable to form hydrogen bonds DNA molecule 1 Phosphate H N N G N N N N N N H H H H H H H O H N N A N N N N N H CH 3 H H H H O T N Phosphate DNA molecule 2 Sugar O

6 DNA-binding motifs Regions of regulatory proteins which bind to DNA –Helix-turn-helix motif Homeodomain motif –Zinc finger motif –Leucine zipper motif

7 3.4 nm a. The Helix-Turn-Helix Motif α Helix Turn 90° α Helix (Recognition helix) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Turn

8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Leucine Zipper Motif Zipper region b. 3.4 nm a. The Helix-Turn-Helix Motif α Helix Turn 90° α Helix (Recognition helix) Turn

9 Prokaryotic regulation Control of transcription initiation –Positive control – increases frequency of initiation of transcription Activators enhance binding of RNA polymerase to promoter Effector molecules can enhance or decrease –Negative control – decreases frequency Repressors bind to operators in DNA Allosterically regulated Respond to effector molecules – enhance or abolish binding to DNA

10 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

11 lac operon Contains genes for the use of lactose as an energy source  -galactosidase (lacZ), permease (lacY), and transacetylase (lacA) Gene for the lac repressor (lacI) is linked to the rest of the lac operon

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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 13

14 Allolactose Z Y A RNA polymerase is not blocked and transcription can occur a. Glucose Low, Inducer Present, Promoter Activated CAP DNA cAMP cAMP–CAP binds to DNA Glucose level is low cAMP is high cAMP activates CAP by causing a conformation change cAMP CAP- binding site Repressor will not bind to DNA mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Y A b. Glucose High, Inducer Absent, Promoter Not Activated Glucose is available cAMP level is low Repressor binds to DNA CAP does not bind Effector site is empty, and there is no conformation change RNA polymerase is blocked by the lac repressor

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 16

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18 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

19 The trp operon is negatively 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

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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 22

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 23

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25 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

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27 Coactivators and mediators are also required for the function of transcription factors –Bind to transcription factors and bind to other parts of the transcription apparatus –Mediators essential to some but not all transcription factors –Number of coactivators is small because used with multiple transcription factors

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 28

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30 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

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 31

Some coactivators have been shown to be histone acetylases Transcription is increased by removing higher order chromatin structure that would prevent transcription “Histone code” postulated to underlie the control of chromatin structure 32

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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 34

35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. Nucleosome sliding 2. Remodeled nucleosome 3. Nucleosome displacement4. Histone replacement ADP + ATP PiPi ATP -dependent remodeling factor

36 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

37 Micro RNA or miRNA Production of a functional miRNA begins in the nucleus Ends in the cytoplasm with a ~22 nt RNA that functions to repress gene expression miRNA loaded into RNA induced silencing complex (RISC) RISC is targeted to repress the expression of genes based on sequence complementarity to the miRNA

38 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cytoplasm RNA Polymerase II microRNA gene Pri-microRNA Nucleus Drosha Exportin 5 Pre-microRNA Dicer Mature miRNA RISC mRNA RISC mRNA cleavage mRNA RISC Inhibition of translation RNA Polymerase II microRNA gene Pri-microRNA Nucleus Drosha Exportin 5 Pre-microRNA

siRNA RNA interference involves the production of siRNAs Production similar to miRNAs but siRNAs arise from long double-stranded RNA Dicer cuts yield multiple siRNAs to load into RISC Target mRNA is cleaved 39

miRNA or siRNA? Biogenesis of both miRNA and siRNA involves cleavage by Dicer and incorporation into a RISC complex Main difference is target –miRNA repress genes different from their origin –Endogenous siRNAs tend to repress genes they were derived from 40

41 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Exogenous dsRNA, transposon, virus P P P P P P P P siRNAs + Ago RISC Ago RISC siRNA in RISC Cleavage of target mRNA Repeated cutting by dicer mRNA

42 Alternative splicing Introns are spliced out of pre-mRNAs to produce the mature mRNA Tissue-specific alternative splicing Same gene makes calcitonin in the thyroid and calcitonin-gene related peptide (CGRP) in the hypothalamus Determined by tissue-specific factors that regulate the processing of the primary transcript

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44 RNA editing Creates mature mRNA that are not truly encoded by the genome Involves chemical modification of a base to change its base-pairing properties Apolipoprotein B exists in 2 isoforms –One isoform is produced by editing the mRNA to create a stop codon –This RNA editing is tissue-specific

45 Initiation of translation can be controlled –Ferritin mRNA only translated if iron present Mature mRNA molecules have various half-lives depending on the gene and the location (tissue) of expression –Target near poly-A tail can cause loss of the tail and destabilization

46 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA 2. RNA splicing Gene expression can be controlled by altering the rate of splicing in eukaryotes. Alternative splicing can produce multiple mRNAs from one gene. Mature RNA transcipt Exons Introns 5´ cap 3´ poly-A tail Cut intron RNA polymerase II 3´3´ 5´5´ Primary RNA transcript 1. Initiation of transcription Most control of gene expression is achieved by regulating the frequency of transcription initiation.

47 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3. Passage through the nuclear membrane Gene expression can be regulated by controlling access to or efficiency of transport channels. 4. Protein synthesis Many proteins take part in the translation process, and regulation of the availability of any of them alters the rate of gene expression by speeding or slowing protein synthesis. 3´3´ 5´5´ Small subunit Nuclear pore 5´ cap mRNA Large subunit 3´ poly-A tail

48 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5. RNA interference Gene expression is regulated by small RNAs. Protein complexes containing siRNA and miRNA target specific mRNAs for destruction or inhibit their translation. 6. Posttranslational modification Phosphorylation or other chemical modifications can alter the activity of a protein after it is produced. Completed polypeptide chain RISC P P

49 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

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