Chapter 16 Lecture Outline See separate PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes and animations. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Control of Gene Expression Chapter 16 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

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

Major groove Minor groove Major groove Minor groove Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Vantage point = Hydrogen bond donors = Hydrogen bond acceptors = Hydrophobic methyl group = Hydrogen atoms unable to form hydrogen bonds DNA molecule 1 Major groove H H N O H N H N G N H N C H Phosphate N N N H O H Minor groove DNA molecule 2 Major groove H H N N H O CH3 Phosphate N A N H N T H N N Sugar H O Minor groove

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

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

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

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

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

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

I P CAP-binding site Gene for repressor protein Operator Gene for Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CAP-binding site Gene for repressor protein Operator Gene for permease Promoter for lac operon Promoter for I gene Genefor ß-galactosidase Gene for transacetylase P I CAP lac O P I Z Y A Regulatory region Coding region lac Control system

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

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

blocked by the lac repressor 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 A Repressor binds to DNA CAP does not bind Y Effector site is empty, and there is no conformation change RNA polymerase is blocked by the lac repressor b.

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

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

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

RNA polymerase is not blocked, and transcription can occur Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Tryptophan Absent, Operon Derepressed E Inactive trp repressor (No tryptophan present) D Translation C B A trp repressor cannot bind to DNA mRNA Enzymes for tryptophan synthesis produced Operator C B A D E Gene for trp repressor Promoter for trp operon RNA polymerase is not blocked, and transcription can occur a.

b. Tryptophan Present, Operon Repressed Tryptophan binds to repressor, Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Tryptophan Present, Operon Repressed Tryptophan binds to repressor, causing a conformation change Tryptophan Repressor conformation change allows it to bind to the operator RNA polymerase is blocked by the trp repressor, and transcription cannot occur B A D C Enzymes for tryptophan synthesis not produced E Gene for trp repressor b.

Tryptophan repressor Tryptophan 3.4 nm Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Tryptophan 3.4 nm

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

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

Interactions of various factors Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA Polymerase II Transcribed region TAFs B F E TFIID H TATA box A Core promoter

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

Courtesy of Dr.Harrison Echols and Dr. Sydney Kustu DNA looping Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NtrC (activator) 5 nm Enhancer Promoter RNA polymerase Bacterial RNA polymerase is loosely bound to the promoter. The activator (NtrC) binds at the enhancer. ATP ADP 5 nm DNA loops around so that the activator comes into contact with the RNA polymerase. RNA polymerase Activator mRNA synthesis The activator triggers RNA polymerase activation, and transcription begins. DNA unloops. Courtesy of Dr.Harrison Echols and Dr. Sydney Kustu

Enhancers RNA polymerase Transcription factor Transcribed Activator Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Promoter Enhancer Activator Transcription factor RNA polymerase Transcribed region TATA box mRNA synthesis

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

Activators General factors B F E RNA polymerase II TFIID H A Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Enhancers Coding region Activator Activators General factors 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. Enhancer Coactivator TAFs B F Activator E RNA polymerase II TFIID 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.

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

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

Amino acid histone tail Condensed solenoid Acetyl group 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 Condensed solenoid 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

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

Pi ADP + ATP ATP-dependent remodeling factor 1. Nucleosome sliding Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ADP + Pi ATP ATP-dependent remodeling factor 1. Nucleosome sliding 2. Remodeled nucleosome 3. Nucleosome displacement 4. Histone replacement

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

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

Copyright © The McGraw-Hill Companies, Inc Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA Polymerase II RNA Polymerase II microRNA gene microRNA gene Pri-microRNA Pri-microRNA Nucleus Nucleus Pre-microRNA Drosha Drosha Pre-microRNA Exportin 5 Exportin 5 Cytoplasm Dicer Mature miRNA Ago Ago RISC mRNA Ago Ago RISC mRNA cleavage Target mRNA Ago Ago Ago Ago RISC RISC 39 Inhibition of translation

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

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

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

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

Primary RNA transcript Introns are spliced Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 1 2 2 3 3 4 4 5 5 6 5´cap 3´ Poly- A tail Primary RNA transcript Introns are spliced Exons Thyroid splicing pattern Hypothalamus splicing pattern Introns 1 2 3 4 1 2 3 5 6 5´cap 3´ Poly- A tail 5´cap 3´ Poly- A tail Mature mRNA Mature mRNA Calcitonin CGRP

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

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

Copyright © The McGraw-Hill Companies, Inc Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA polymerase II 1. Initiation of transcription . Transcription is controlled by the frequency of initiation. This involves transcription factors that bind to promoters and enhancers. 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. Cut intron DNA 3´ 3´poly-A tail 5´cap 5´ Primary RNA transcript Exons Mature RNA transcipt Introns

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

Ubiquitin Protein Proteasome Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ubiquitin 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. Protein degradation. Proteins to be degraded are labeled with ubiquitin, then destroyed by the proteasome. Protein RISC Proteasome

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

Pi Ubiquitin Protein to be destroyed ATP ADP + Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ubiquitin Protein to be destroyed ATP Pi ADP + Destroyed by proteolysis

Polypeptide fragments Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Degradation Polypeptide fragments Proteasome ATP ADP + Pi Ubiquitination Ubiquitin ADP + Pi ATP ATP Targeted protein ADP Ubiquitin ligase