Regulation of Gene Expression

28 | Regulation of Gene Expression 2017 W. H. Freeman and Company CHAPTER 28 Regulation of Gene Expression Learning goals DNA elements that control transcription Protein factors that control transcription Lac operon as a model for regulation Regulation of protein synthesis by RNA Ways to Regulate Protein

Concentration in a Cell Synthesis of primary RNA transcript How to process this RNA into mRNA Posttranscriptional modifications of mRNA Degradation of mRNA Protein synthesis Posttranslational modification of protein Targeting and transport of the protein Degradation of the protein Seven Processes That Affect the SteadyState Concentration of a Protein Trends in Understanding Gene

Regulation Past focus has been on understanding transcription initiation. There is increasing elucidation of posttranscriptional and translational regulation. Mechanisms can be elaborate and interdependent, especially in development. Regulation relies on precise protein-DNA and protein-protein contacts. The Vocabulary of Gene Regulation Housekeeping gene under constitutive expression

constantly expressed in approximately all cells Regulated gene Levels of the gene product rise and fall with the needs of the organism. Such genes are inducible. able to be turned on Such genes are also repressible. able to be turned off RNA Polymerase Binding to Promoters Is a Major Target of Regulation

RNA polymerases bind to promoter sequences near the starting point of transcription initiation. The RNA pol-promoter interaction greatly influences the rate of transcription initiation. Regulatory proteins (transcription factors) work to enhance or inhibit this interaction between RNA pol and the promoter DNA. A Consensus Sequence Is Found in Many E. Coli Promoters Most bacterial promoters include the conserved 10 and 35 regions that interact with the factor of RNA polymerase.

Substitutions in this 10 to 35 region usually reduce the affinity of RNA Pol for the promoter. Some promoters also include the upstream element that interacts with the subunit of RNA polymerase. Mechanisms to Regulate Transcription in Bacteria Use of factors recognize different classes of promoters allows coordinated expression of different sets of genes Binding other proteins (transcription factors) to promoters

recognize promoters of specific genes may bind small signaling molecules may undergo posttranslational modifications proteins affinity toward DNA is altered by ligand binding or posttranslational modifications allows expression of specific genes in response to signals in the environment

Regulation by Specificity Factors Such as Subunits of RNA Pol Specificity factors alter RNA polymerases affinity for certain promoters. Example: subunit of E. coli RNA Pol Most E. coli promoters recognized by 70. This subunit can be replaced by one of six additional specificity factors. Heat shock will replace 70 with 32 and direct RNA Pol to different promoters. Heat Shock Induces Transcription of New Products to Protect Cell

Occurs when bacteria are subject to heat stress RNA Pol replaces 70 with 32 Causes RNA Pol to bind to different set of promoters transcription of new products including chaperones that keep proteins in correct conformation, even in heat Small-Molecule Effectors Can Regulate Activators and Repressors Repressors reduce RNA Pol-promoter interactions or block the polymerase. bind to operator sequences on DNA usually near a promoter in bacteria but further away in many eukaryotes

Effectors can bind to repressor and induce a conformational change. change may increase or decrease repressors affinity for the operator and thus may increase or decrease transcription Activators Improve Contacts Between RNA Polymerase and the Promoter Binding sites in DNA for activators are called enhancers. In bacteria, enhancers are usually adjacent to the promoter.

often adjacent to promoters that are weak (bind RNA polymerase weakly), so the activator is necessary In eukaryotes, enhancers may be very distant from the promoter. Negative Regulation Negative regulation involves repressors. Example: Repressor binds to DNA and shuts down transcription Alternative: Signal causes repressor to dissociate from DNA; transcription induced

Despite opposite effects on transcription, both are negative regulation Positive Regulation Positive regulation involves activators. Enhance activity of RNA polymerase Activator-binding sites are near promoters that weakly bind RNA Pol or do not bind at all. It may remain bound until a molecule signals

dissociation. Alternatively, the activator may only bind when signaled. DNA Looping Allows Eukaryotic Enhancers to Be Far from Promoters Activators can influence transcription at promoters thousands of bp away. How? Via formation of DNA loops Looping can be facilitated by

architectural regulator proteins. Co-activators may mediate binding by binding to both activator and RNA polymerase. Many Bacterial Genes Are Transcribed And Regulated Together in an Operon An operon is a cluster of genes sharing a promoter and regulatory sequences. Genes are transcribed together, so mRNAs are several genes represented on one mRNA (polycistronic).

First example: the lac operon The lac Operon Reveals Many Principles of Gene Regulation Work of Jacob and Monod 1960 Shows how three genes for metabolism of lactose are regulated together as an operon: -galactosidase (lacZ) cleaves lactose to yield glucose and galactose lactose permease (galactoside permease; lacY) transports lactose into cell

thiogalactoside transacetylase (lacA) Thet rely on negative regulation via a repressor. Lactose Metabolism in E. Coli When glucose is abundant and lactose is lacking, cells make only very low levels of enzymes for lactose metabolism. Transcription is repressed. If glucose is scarce and cells are fed lactose, the cells can use it as their energy source. The cells suddenly express the genes for the

enzymes for lactose metabolism. Transcription is no longer repressed. Lactose Metabolism in E. Coli Inhibiting the Transcription of the lac Operon via a Repressor Protein A gene called lacI encodes a repressor called the Lac repressor. It has its own promoter PI. Transcription of the repressor is independent of transcription of the enzymes the repressor regulates. The repressor can bind to three operator sites (O1O3).

The Lac repressor binds primarily to the operator O1. O1 is adjacent to the promoter. Binding of the repressor helps prevent RNA polymerase from binding to the promoter. The repressor also binds to one of two secondary operators, with the DNA looped between this secondary operator and O1 (see Fig. 28-8b). It reduces transcription, but transcription occurs at a low, basal rate, even with the repressor bound. Structure of the lac Operon

Lac Repressor Bound to O1 and O3 with DNA Looped Between The lac Operon Is Induced by Allolactose Allolactose (an inducer) binds to the repressor and causes it to dissociate from the operator. -galactosidase not only hydrolyzes lactose, but it can also isomerize lactose into allolactose. [Allolactose] when [Lactose] How Lac Repressor Binds to DNA

Lac repressor is a tetramer. dimer of dimers Each dimer binds to the palindromic operator sequence. ~1722 bp of contact Kd ~1010 M The O1 sequence reflects the symmetry of the repressor. There are approximately 20 repressors per cell. The lac Operon Is Governed by More Than Repressor Binding

The availability of glucose governs expression of lactose-digesting genes via catabolite repression. When glucose is present, lactose genes are turned off. It is mediated by cAMP and cAMP receptor protein (CRP or CAP for catabolite activator protein). When Glucose Is Absent, lac Operon Transcription Is Stimulated by CRP-cAMP cAMP binds near the promoter. stimulates transcription 50fold

bends DNA open complex doesnt form readily without CRP-cAMP CRP-cAMP only has this effect when the Lac repressor has dissociated. cAMP is made when [glucose] is low. When Lactose Is Absent Little to No Transcription Occurs Whether [glucose] is high or low, if lactose is absent

repressor stays bound. no transcription even when CRP-cAMP bind. When Lactose Is Present, Transcription Depends On Glucose Level Repressor dissociates, but transcription is only stimulated significantly if cAMP rises. Two Requirements for Strongest Induction of the lac Operon 1. Lactose must be present to form allolactose to bind to the repressor and cause it to dissociate from the operator.

reducing repression 2. [Glucose] must be low so that cAMP can increase, bind to CRP, and the complex can bind near the promoter causing activation Combined Effects of Glucose and Lactose on the lac Operon When lactose is low, repressor is bound: inhibition When lactose is high, repressor dissociates permitting transcription When glucose is high, CRP is not bound and

transcription is dampened When glucose is low, cAMP is high and CRP is bound activation Binding of Proteins to DNA Often Involves Hydrogen Bonding Gln/Asn can form a specific H-bond with adenines N-6 and H-7 Hs. Arg can form specific H-bonds with the cytosine-guanine base pair. See Fiure. 28-10. The major groove is the right size for the helix and has exposed H-bonding groups.

Gln/Asn + Adenine and Arg + C-G Protein-DNA Binding Motifs A few protein arrangements are used in thousands of different regulatory proteins and are hence called motifs. helix-turn-helix used by Lac repressor zinc finger leucine zipper

and so on The Helix-Turn-Helix Motif Is Common in DNA-Binding Proteins ~ 20 aa one helix for recognition for DNA (red in the next slide), then turn, then another helix sequence-specific binding due to specific contacts between the recognition helix and the major groove Four DNA-binding helix-turn-helix motifs (gray) in the Lac repressor

Helix-Turn-Helix Motif The Zinc Finger Motif Is Common in Eukaryotic Transcription Factors ~30 aa Finger portion is a peptide loop cross-linked by Zn2+ Zn2+ usually coordinated by 4 Cys, or 2 Cys, 2 His Interact with DNA or RNA Binding is weak, so several zinc fingers often act in

tandem. Binding can range from sequence specific to random. Zinc Fingers The Leucine Zipper Motif Dimer of two amphipathic helices plus a DNA-binding domain Each helix is hydrophobic on one side and hydrophilic on the other. The hydrophobic side is the contact between the two monomers.

Approximately every seventh residue in helices is Leu. Helices form a coiled coil. The DNA-binding domain has basic residues (Lys, Arg) to interact with polyanionic DNA. Structure of a Leucine Zipper: GCN4 from Yeast Eukaryotic Gene Regulation Relies on Combinatorial Control In yeast, there are only 300 transcription factors for thousands of genes. Transcription factors mix and match.

Different combinations regulate different genes. Eukaryotic gene regulation relies on proteinprotein interactions. Eukaryotic RNA-Binding Domain RNA recognition motifs (RRMs) 90100 amino acids four strand antiparallel sheet with two helices Found in gene activators and bind to both DNA and RNA DNA binding induces transcription. Binding to lncRNAs (long noncoding RNAs) forces the proteins to compete with DNA for binding, thus decreasing gene transcription. They may bind to mRNA, rRNA, or other ncRNAs.

This motif may be part of DNA-binding regulatory proteins or may occur in proteins binding only RNA. Eukaryotic RRM Binding to DNA and RNA Amino Acid Biosynthesis Regulated by Transcriptional Attenuation Bacterial operons are also found for biosynthetic pathways. The trp operon is regulated by transcription attenuation. Transcription begins but is then halted by a stop signal

(attenuator). It relies on the fact that, in bacteria, transcription and translation can proceed simultaneously. The attenuator sequence is in the 5-region of a leader sequence, and it can make the ribosome stall. Role of the Attenuator The attenuator (purple, next slide), which is part of the leader (light blue) determines: if transcription will be attenuated at the end of the leader or, if transcription will continue into the genes for Trp synthesis

The trp Operon The Leader Region Can Form Different Stem-Loop Structures The leader is 162 nucleotides long. includes segments 14 If segments 3 and 4 base-pair, they form a hairpin structure that is the attenuation signal. If segments 2 and 3 base-pair, transcription proceeds and the trp synthetic enzymes are made.

no attenuation The Four Segments of the Trp Leader Region The 34 Pair (Attenuator) and the 23 Pair Abundance of tRNATrp Leads to Formation of the Attenuator Segment 1 is transcribed and immediately translated. The ribosome is close behind RNA Pol. Segment 1 contains important Trp codons.

If tRNATrp is abundant, translation proceeds so that segment 2 is covered with the ribosome and cant pair with segment 3. so segment 3 pairs with 4 attenuator Low Availability of tRNATrp Signals Translation to Continue If tRNATrp is scarce, the ribosome will stall at the Trp codons in the mRNA. allows 23 pairs to form Translation proceeds unhindered.

Other amino acid synthesis operons also use this regulation mechanism (e.g., Leu, His, Phe). Trp Operon When Trp Synthesis Is Not Needed (tRNATrp Is High) Trp Operon When Trp Levels Are Low, tRNATrp Not Abundant, and Trp Synthesis Is Needed A Repressor Protein Also Regulates TRP Transcription The Trp operon also has a repressor that binds

to DNA in the presence of tryptophan. Trp repressor is a homodimer. When Trp is abundant, it binds to repressor, causes it to bind to the operator, and slows expression of genes for Trp synthesis. It has helix-turn-helix motifs that interact with DNA via the major groove. Regulation of the SOS Response SOS Response = response to extensive DNA damage results in cell cycle arrest and activation of DNA repair systems

Normally, SOS genes are repressed by LexA repressor. LexA binds to operators at several genes. Damaged DNA produces a lot of single strands. ssDNA is bound by the protein RecA (or, in eukaryotes Rad51). activates RecAs ability to interact with LexA repressor RecA binds to LexA repressor, causing it to self-cleave and dissociate from DNA. RecA is called a co-protease. Regulation of the SOS Response

in E. Coli Link Between the SOS Response and Virus Propagation Some repressors keep viruses in a dormant state within the bacterial host. RecA (Rad51 in eukaryotes) can help cleave and inactivate these other repressors. allows virus to replicate, lyse cell, and release new virus particles Synthesis of Ribosomal Proteins and rRNA is Controlled at Translation

When bacteria need more protein (as in cell growth), they make more ribosomes. Ribosome synthesis consumes energy and resources, so it is highly regulated. Ribosmal protein (r-protein) operons are regulated via translational feedback (next slide). Translational Feedback Mechanism Each operon for an r-protein encodes a translational repressor. repressor binds to mRNA and blocks translation

Repressor has greater affinity for rRNA than for mRNA. so translation is repressed only when synthesis of r-proteins exceeds a level needed to make ribosomes Translational Feedback in Ribosomal Protein Operons rRNA Synthesis Is Also Regulated by Amino Acid Availability The stringent response occurs when aa

concentrations are low. Lack of aa produces uncharged tRNA. Uncharged tRNA binds to ribosomal A site. rRNA synthesis triggers a cascade that begins with binding stringent factor protein (RelA) to ribosome. Stringent Factor Catalyzes Formation of an Unusual Guanosine-Based Messenger Stringent factor catalyzes formation of nucleotide guanosine tetraphosphate (ppGpp). It is formed from adding diphosphate (pyrophosphate) to the 3-end of GTP.

Then a phosphorylase cleaves a phosphate to yield ppGpp. Binding of ppGpp to RNA polymerase reduces rRNA synthesis. Stringent Response in E. Coli Some RNAs Participate in Regulation Cis regulation: a molecule affects its own function Trans regualtion: a molecule is affected by

another separate molecule Example: mRNA of gene rpoS (RNA polymerase sigma factor) that encodes S, a specificity factor used by E. coli in stress conditions such as starvation when S needed to transcribe stress response genes Trans-Acting sRNAs Facilitate Translation of S mRNA S mRNA is present a low levels but is not translated due to a hairpin structure that inhibits its binding to ribosomes. sRNAs (small RNAs) bind to this mRNA and

inhibit formation of the hairpin . These sRNAs include DsrA and RprA. sRNA-mRNA interactions are facilitated by a chaperone protein called Hfq. Regulation of Bacterial mRNA Function in Trans by sRNA Activation of Bacterial Translation by Small RNA Molecules The ribosome-binding ShineDalgarno sequence is sequestered into a stem-loop structure in the mRNA.

In the presence of protein Hfq, small regulatory RNA DsrA binds to the mRNA. The binding of DsrA opens up the stem-loop and allows mRNA binding to the ribosome. DsrA RNA promotes translation. Inhibition of Bacterial Translation by Small RNA Molecules The ribosome-binding ShineDalgarno sequence is sequestered into a stem-loop structure in the mRNA. In the presence of protein Hfq, small regulatory RNA OxyS binds to the mRNA. The binding of OxyS blocks the ribosome binding site

in mRNA. OxyS RNA inhibits translation. Cis Regulation by Riboswitches Riboswitch = domain of an mRNA that can bind a smallmolecule ligand The binding of ligand affects conformation of the mRNA and its activity. Thus, riboswitches allow mRNA to participate in their own regulation and respond to changing concentrations of

the ligand. Riboswitches Are a Developing Area of Research Riboswitches have been found to respond to many coenzymes, metabolites, and so on. They are also found in eukaryotic introns and seem to regulate splicing. Some riboswitches are unique to bacteria and are therefore a target for antibiotics. Regulation by Gene Recombination Processes can remove promoters

relative to the coding sequence or can put genes into multiple orientations to alter the expression. Example: Flagellin genes in Salmonella In one orientation fljB is expressed with a repressor for fljC gene. In another orientation, only fljC is expressed. The process is called phase variation. Phase Variation Regulation of Flagellin Genes

TABLE 28-1 Examples of Gene Regulation by Recombination System Recombinase/ recombination site Type of recombination

Function Phase variation (Salmonella) Hin/hix Site-specific Alternative expression of two flagellin genes allows evasion of host immune response. Host range (bacteriophage )

Gin/gix Site-specific Alternative expression of two sets of tail fiber genes affects host range. Mating-type switch (yeast) HO endonuclease, RAD52 protein, other proteins/MAT

Nonreciprocal gene conversiona Alternative expression of two mating types of yeast, a and , creates cells of different mating types that can mate and undergo meiosis. Nonreciprocal gene conversiona Successive expression of different

genes encoding the variable surface glycoproteins (VSGs) allows evasion of host immune response. Antigenic variation (trypanosomes)b Varies In nonreciprocal gene conversion (a class of recombination events not discussed in Chapter 25), genetic information is moved from one part of the genome (where it is silent) to another (where it is expressed). The reaction is similar to replicative transposition (see Fig. 25-42). b

Trypanosomes cause African sleeping sickness and other diseases (see Box 6-3). The outer surface of a trypanosome is made up of multiple copies of a single VSG, the major surface antigen. A cell can change surface antigens to more than 100 different forms, precluding an effective defense by the host immune system. Function a Features of Eukaryotic Gene Regulation Access of eukaryotic promoters to RNA polymerase is hindered by chromatin structure. thus requires remodeling chromatin Positive regulation mechanisms predominate and are required for even a basal level of gene

expression. Eukaryotic gene expression requires a complicated set of proteins. Three Features of Transcriptionally Active Chromatin Euchromatin = less-condensed chromatin, distinguished from transcriptionally inactive heterochromatin Chromatin remodeling of transcriptionally active genes: nucleosomes repositioned histone variants

covalent modifications to nucleosomes Nucleosomes Can Be Restructured by Specific Protein Complexes SWI/SNF (SWItch/Sucrose NonFermentable) complex remodels chromatin to irregularly space nucleosomes stimulates binding of transcription factors works with proteins of ISWI (imitation switch) family ATP-dependent alteration of spacing between nucleosomes, and so on

Nucleosome Remolding Eukaryotic cells generally have 910 different CHD (chromodomain helicase DNA-binding) grouped into three subfamilies. Each family has a specialized role. Ie: INO80 family: roles in remodeling chromatin and DNA repair SWR1 promotes subunit exchange in nucleosomes inducing histone variants Ie: H2Az found in transcriptionally active regions Covalent Modification of Histones

Methylation Phosphorylation Acetylation Ubiquitination Sumoylation Occur mostly in the N-terminal domain of

the histones found near the exterior of the nucleosome particle Histone Modification Alters Transcription Covalent modification of histones allows recruitment of enzymes and transcription factors. Methylation of Lys-4 and Lys-36 at histone3 (H3) and Arg of H3 and H4: results in transcriptional activation recruits histone acetyltransferases (HATs) that then acetylate a particular Lys reversed by histone deacetylases (HDACs) that make

chromatin inactive Acetylation of Lys results in decreased affinity of histone for DNA. TABLE 28-2 Enzyme complexa Some Enzyme Complexes That Catalyze Chromatin Structural Changes Associated with Transcription Oligomeric structure (number of polypeptides)

Source Activities 817 Mr > 106 Eukaryotes Nucleosome remodeling; transcriptional activation ISWI family

24 Eukaryotes Nucleosome remodeling; transcriptional repression; transcriptional activation in some cases CHD family 110 Eukaryotes

Nucleosome remodeling; nucleosome ejection for transcriptional activation; some have repressive roles INO80 family >10 Eukaryotes Nucleosome remodeling and transcriptional activation; family member SWR1 engages in replacement of H2A-H2B with H2AZ-H2B

Yeast GCN5 has type A HAT activity Histone movement, replacement, or editing, requiring ATP SWI/SNF family Histone modification GCN5-ADA2-ADA3 3

SAGA/PCAF >20 Eukaryotes Includes GCN5-ADA2-ADA3; acetylates residues in H3, H2B, H2AZ NuA4 12

Eukaryotes EsaI component has HAT activity; acetylates H4, H2A, and H2AZ 1 Eukaryotes Eukaryotes Deposition of H3.3 during transcription Histone chaperones not requiring ATP HIRA

The abbreviations for eukaryotic genes and proteins are often more confusing or obscure than those used for bacteria. SWI (switching) was discovered as a protein required for expression of certain genes involved in mating-type switching in yeast, and SNF (sucrose nonfermenting) as a factor for expression of the yeast gene for sucrase. Subsequent studies revealed multiple SWI and SNF proteins that act in a complex. The SWI/SNF complex has a role in expression of a wide range of genes and has been found in many eukaryotes, including humans. ISWI is imitation SWI. CHD is chromodomain, helicase, DNA binding; INO80, inositol-requiring 80; and SWR1, SWi2/Snf2-related ATPase 1. The complex of GCN5 (general control nonderepressible) and ADA (alteration/deficiency activation) proteins was discovered during investigation of the regulation of nitrogen metabolism genes in yeast. These proteins can be part of the larger SAGA (SPF, ADA2,3, GCN5, acetyltransferase) complex in yeasts. The equivalent of SAGA in humans is PCAF (p300/CBP-associated factor). NuA4 is nucleosome acetyltransferase of H4; ESA1, essential SAS2related acetyltransferase. HIRA is histone regulator A. a Idea of a Nucleosome Code or Histone Code Some speculate that genomes encode

directions for nucleosome organization. Nucleosomes seem to occur at specific sequences. Covalent modifications seem to occur at specific regions/sequences. A nucleosome positioning code or histone modification code explains these observations. Positive Regulation of Eukaryotic Promoters Eukaryotic gene transcription is usually dependent on activator proteins, not RNA Pol affinity. Most promoters are inaccessible, thus making

repressors redundant. Combinatorial control provides a more precise positive control for gene regulation. Negative regulation exists but typically involves lncRNAs not proteins. RNA Polymerase II Requires Five Types of Proteins Transcription activators (enhancers) proteins that bind to upstream activator sequences (UASs) Architectural regulators to facilitate DNA looping Chromatin modification/remodeling proteins

Coactivators act indirectly (with other proteins, not with DNA) Basal (general) transcription factors Enhancer Proteins Are Diverse Can bind thousands of nucleotides away from the TATA box of the promoter Can have DNA-binding, protein-binding, and/or signal molecule-binding domains can bind with multiple proteins Some regulate a few genes; some regulate

many hundreds of genes Architectural Regulators Regulate Looping Looping of DNA allows distant enhancers to modulate assembly at promoters. Example: high mobility group (HMG) proteins have multiple

functions, including architectural regulation Coactivators Assist RNA Polymerase Mediator complex binds to carboxyl-terminal domain(CTD) of RNA Pol II required for both basal and regulated transcription at many promoters later provides assembly surface for other complexes

TATA-binding protein is first component of preinitiation complex (PIC) at the typical TATA box of a promoter Details of Eukaryotic Regulation That Are Emerging Binding of activators triggers many promoters to bind RNA Pol II. Binding one activator seems to enable binding of additional activators. Often, components bind in a regular order.

Histones are displaced as activators bind. The process coordinates with chromatin remodeling. Yeast Galactose Metabolism The regulation of genes for importing and metabolizing galactose in yeast illustrates important principles. Genes for galactose metabolism (GAL) are spread over several chromosomes. But all have similar promoters. TATA box, Inr sequences, upstream activator sequence (UASG) recognized by Gal4 protein (Gal4p)

All GAL Genes Are Regulated by a Common Set of Proteins Galactose binds Gal3p, then forms a complex with Gal4p and Gal80p. It allows Gal4p to be activator. Gal4 also recruits the SWI/NSF and mediator complexes for opening the chromatin. TABLE 28-3 Gene

Genes of Galactose Metabolism in Yeast Protein function Relative protein expression in different carbon sources Chromosomal location Protein size (number of residues)

Glucose Glycerol Galactose II 528

+++ Regulated genes GAL1 Galactokinase GAL2 Galactose permease

XII 574 +++ PGM2 Phosphoglucomutase

XIII 569 + + ++ GAL7

Galactose 1-phosphate uridylyltransferase II 365 +++

GAL10 UDP-glucose 4-epimerase II 699 +++

MEL1 -Galactosidase II 453 +

++ IV 520 + ++ Regulatory Genes

GAL3 Inducer GAL4 Transcriptional activator XVI 881 +/

+ + GAL80 Transcriptional inhibitor XIII 435

+ + ++ Source: Information from R. Reece and A. Platt, Bioessays 19:1001, 1997. Features of Hormone-Mediated Regulation Hormone-receptor complex binds to DNA regions called hormone response elements (HREs).

Hormone receptors have a DNA-binding domain with zinc fingers. Hormone receptors also have a ligand-binding region at the C-terminus that is highly variable between different receptors. Two Types of Hormone Receptors Monomeric Type 1 (NR)receptors for sex hormones and glucocorticoids found in cytoplasm in complex with Hsp70 when hormone binds, Hsp70

dissociates receptor dimerizes exposes nuclear localization region so hormone-receptor complex travels to nucleus to be transcriptional activator Two Types of Hormone Receptors Type II (includes thyroid hormone receptor; TR) found in nucleus bound

to DNA and corepressor retinoid X receptor (RXR) hormone binds, corepressor dissociates receptor-hormone complex then activates transcription Eukaryotic Gene Expression Is Also Regulated by Peptide Hormones 2 messengers lead to activation of transcription factors

Example: -adrenergic pathway cAMP activates protein kinase A (PKA). PKA enters the nucleus and phosphorylates cAMP response element-binding protein (CREB). CREB is a transcription activator of genes leading to fuel use, rather than fuel storage. Four Mechanisms of Translation Regulation 1. Phosphorylation of translation initiation factors 2. Translational repressors (typically bind to 3 UTR)

3. Disruption of eIF4E and eIF4G interactions 4. RNA-mediated regulation (gene silencing) Micro-RNAs Prevent Translation of mRNA Micro-RNAs (miRNAs) silence genes by binding to mRNAs can prevent transcription of the mRNA by cleaving it (via endonucleases Drosha or Dicer) or by blocking it Some miRNAs are made briefly during development; they are called small temporal

RNAs (stRNAs). Researchers Can Shut Down Genes Artificially via RNA Interference Any dsRNA that corresponds to an mRNA and is introduced into a cell will be cleaved by Dicer into short segments called small interfering RNAs (siRNAs). These will bind to the mRNA to silence its translation. The process is called RNA interference. Gene Silencing by RNA Interference

ncRNAs Also Regulate Gene Expression Referred to as lncRNAs when longer than 200 nt example: HSR1 lncRNA (~600nts) interacts with eEF1a to regulate translation in stress cells example: 7SK binds to RNA Pol II transcription factor pTEFb to repress elongation Our knowledge of ncRNAs is rapidly expanding as further research continues to explore this field.

Regulatory Proteins Control Development Embryonic development requires complex and intricate coordination and regulation of gene expression. More genes are expressed during early development than any other part of the life cycle in a differentiated tissue. example: sea urchin with ~18,500 different mRNAs in oocyte versus ~6,000 different mRNAs in a differentiated tissue

Regulatory Proteins Control Development Polarity must be established (anterior vs. posterior and dorsal vs. ventral). The embryo body may have repeating segments (Drosophila) (metamerism) with characteristic features. Morphogens, proteins that cause surrounding tissues to change shape, affect development. Genes from both mother (maternal genes) and embryo affect development and proper segmentation. Homeotic versus Maternal Genes

Homeotic genes specify development of organs and appendages in a particular body segment Hox genes homeobox contains a conserved sequence present in all of these genes Maternal genes genes expressed in nurse and follicle cells that affect initial polarity and axes development (bicoid and nanos) Regulatory Proteins Control Development Bicoid a transcription factor that activates

expression of several segmentation genes; translational repressor that inactivated certain mRNAs deposited at the anterior pole Lack of Bicoid results in an embryo without a head or thorax and two abdomens. Nanos translational repressor protein deposited at posterior of the egg Translational Regulation of Anterior-Posterior Development Homeotic Genes

Often found in clusters and are highly conserved amount animals Developmental Potential of Stem Cells Stem cells have the potential to differentiate into various tissues. Totipotent cells have the ability to differentiate into ANY tissue type or even a complete organism. Pluripotent cells are able to differentiate into any of the three germ cell layers and many types of tissues, but they cannot make a complete organism.

Unipotent cells can differentiate into only one type of cell or tissue. Developmental Potential of Stem Cells Embryonic stem cells pluripotent cells of the blastocyst used in embryonic stem cell research Adult stem cells limited potential compared with embryonic hematopoietic stem cells can give rise to different cells in niche or microenvironment

considered multipotent example: bone marrow hematopoietic stem cells give rise to many types of blood cells and those that can regenerate bone, but not liver, kidney, and so on Developmental Potential of Stem Cells Chapter 28: Summary In this chapter, we learned that: positive and negative gene regulation occurs in both prokaryotic and eukaryotic organisms there are many useful examples of gene regulation lac

operon, trp operon, and so on regulator proteins contain domains that interact with DNA, RNA, small molecules, and so on ncRNAs are involved in regulation of gene expression eukaryotic gene regulation is complex and involves chromatin remodeling, combinatory control, intercellular and intracellular signals, and gene silencing development in animals is controlled by cascades of regulatory proteins and their precise position within the embryo

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