28 | Regulation of Gene Expression - PowerPoint Presentation

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28 | Regulation of Gene Expression

© 2017 W. H. Freeman and Company

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CHAPTER 28 Regulation of Gene Expression

DNA elements that control transcriptionProtein factors that control transcriptionLac operon as a model for regulationRegulation of protein synthesis by RNA

Learning goals

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Ways to Regulate Protein Concentration in a Cell

Synthesis of primary RNA transcript How to process this RNA into mRNAPosttranscriptional modifications of mRNADegradation of mRNAProtein synthesisPosttranslational modification of proteinTargeting and transport of the proteinDegradation of the protein

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Seven Processes That Affect the Steady-State Concentration of a Protein

Slide5

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.

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The Vocabulary of Gene Regulation

Housekeeping gene under constitutive expressionconstantly expressed in approximately all cellsRegulated geneLevels 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

Slide7

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.

Slide8

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.

Slide9

Mechanisms to Regulate Transcription in Bacteria

Use of σ factors recognize different classes of promotersallows coordinated expression of different sets of genesBinding other proteins (transcription factors) to promotersrecognize promoters of specific genesmay bind small signaling moleculesmay undergo posttranslational modificationsprotein’s affinity toward DNA is altered by ligand binding or posttranslational modificationsallows expression of specific genes in response to signals in the environment

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Regulation by Specificity Factors Such as  Subunits of RNA Pol

Specificity factors alter RNA polymerase’s affinity for certain promoters.Example:  subunit of E. coli RNA PolMost 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.

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Heat Shock Induces Transcription of New Products to Protect Cell

Occurs when bacteria are subject to heat stressRNA Pol replaces 70 with 32Causes RNA Pol to bind to different set of promoters transcription of new products including chaperones that keep proteins in correct conformation, even in heat

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Small-Molecule Effectors Can Regulate Activators and Repressors

Repressors reduce RNA Pol-promoter interactions or block the polymerase.bind to operator sequences on DNAusually near a promoter in bacteria but further away in many eukaryotesEffectors can bind to repressor and induce a conformational change.change may increase or decrease repressor’s affinity for the operator and thus may increase or decrease transcription

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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 necessaryIn eukaryotes, enhancers may be very distant from the promoter.

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Negative Regulation

Negative regulation involves

repressors.Example: Repressor binds to DNA and shuts down transcriptionAlternative: Signal causes repressor to dissociate from DNA; transcription induced

Despite opposite effects on transcription, both are negative regulation

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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.

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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 loopsLooping can be facilitated by architectural regulator proteins.Co-activators may mediate binding by binding to both activator and RNA polymerase.

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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

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The lac Operon Reveals Many Principles of Gene Regulation

Work of Jacob and Monod − 1960Shows how three genes for metabolism of lactose are regulated together as an operon:-galactosidase (lacZ)cleaves lactose to yield glucose and galactoselactose permease (galactoside permease; lacY)transports lactose into cellthiogalactoside transacetylase (lacA)Thet rely on negative regulation via a repressor.

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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.

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Lactose Metabolism in E. Coli

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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 (O1–O3).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.

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Structure of the lac Operon

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Lac Repressor Bound to O1 and O3 with DNA Looped Between

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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] 

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How Lac Repressor Binds to DNA

Lac repressor is a tetramer.dimer of dimersEach dimer binds to the palindromic operator sequence.~17−22 bp of contact Kd ~10−10 MThe O1 sequence reflects the symmetry of the repressor.There are approximately 20 repressors per cell.

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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).

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When Glucose Is Absent,

lac Operon Transcription Is Stimulated by CRP-cAMP

cAMP binds near the promoter.stimulates transcription 50-foldbends DNAopen complex doesn’t form readily without CRP-cAMPCRP-cAMP only has this effect when the Lac repressor has dissociated.cAMP is made when [glucose] is low.

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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.

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When Lactose Is Present, Transcription Depends On Glucose Level

Repressor dissociates, but transcription is only stimulated significantly if cAMP rises.

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Two Requirements for Strongest Induction of the lac Operon

Lactose must be present to form allolactose to bind to the repressor and cause it to dissociate from the operator.reducing repression[Glucose] must be low so that cAMP can increase, bind to CRP, and the complex can bind near the promotercausing activation

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Combined Effects of Glucose and Lactose on the lac Operon

When lactose is low, repressor is bound: inhibitionWhen lactose is high, repressor dissociates permitting transcriptionWhen glucose is high, CRP is not bound and transcription is dampenedWhen glucose is low, cAMP is high and CRP is bound activation

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Binding of Proteins to DNA Often Involves Hydrogen Bonding

Gln/Asn can form a specific H-bond with adenine’s N-6 and H-7 H’s.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.

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Gln/Asn + Adenine andArg + C-G

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Protein-DNA Binding Motifs

A few protein arrangements are used in thousands of different regulatory proteins and are hence called motifs.helix-turn-helixused by Lac repressorzinc fingerleucine zipperand so on

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The Helix-Turn-Helix Motif Is Common in DNA-Binding Proteins

~ 20 aaone  helix for recognition for DNA (red in the next slide), then  turn, then another  helixsequence-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

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Helix-Turn-Helix Motif

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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 HisInteract with DNA or RNA Binding is weak, so several zinc fingers often act in tandem.Binding can range from sequence specific to random.

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Zinc Fingers

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The Leucine Zipper Motif

Dimer of two amphipathic  helices plus a DNA-binding domainEach 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.

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Structure of a Leucine Zipper: GCN4 from Yeast

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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 protein-protein interactions.

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Eukaryotic RNA-Binding Domain

RNA recognition motifs – (RRMs) 90−100 amino acids – four strand antiparallel β sheet with two α helicesFound in gene activators and bind to both DNA and RNADNA 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.

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Eukaryotic RRM Binding to DNA and RNA

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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.

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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

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The trp Operon

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The Leader Region Can Form Different Stem-Loop Structures

The leader is 162 nucleotides long.includes segments 1−4If 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

Slide48

The Four Segments of the Trp Leader Region

Slide49

The 3–4 Pair (Attenuator) and the 2–3 Pair

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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 can’t pair with segment 3. so segment 3 pairs with 4  attenuator

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Low Availability of tRNATrp Signals Translation to Continue

If tRNATrp is scarce, the ribosome will stall at the Trp codons in the mRNA.  allows 2–3 pairs to form Translation proceeds unhindered.Other amino acid synthesis operons also use this regulation mechanism (e.g., Leu, His, Phe).

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Trp Operon When Trp Synthesis Is Not Needed (tRNATrp Is High)

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Trp Operon When Trp Levels Are Low, tRNATrp Not Abundant, and Trp Synthesis Is Needed

Slide54

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.

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Regulation of the SOS Response

SOS Response = response to extensive DNA damageresults in cell cycle arrest and activation of DNA repair systemsNormally, 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 RecA’s ability to interact with LexA repressorRecA binds to LexA repressor, causing it to self-cleave and dissociate from DNA.RecA is called a co-protease.

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Regulation of the SOS Response in E. Coli

Slide57

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

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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).

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Translational Feedback Mechanism

Each operon for an r-protein encodes a translational repressor.repressor binds to mRNA and blocks translationRepressor 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

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Translational Feedback in Ribosomal Protein Operons

Slide61

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.

Slide62

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.

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Stringent Response in E. Coli

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Some RNAs Participate in Regulation

“Cis” regulation: a molecule affects its own function“Trans” regualtion: a molecule is affected by another separate moleculeExample: mRNA of gene rpoS (RNA polymerase sigma factor) that encodes S, a specificity factor used by E. coli in stress conditionssuch as starvation when S needed to transcribe stress response genes

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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.

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Regulation of Bacterial mRNA Function in Trans by sRNA

Slide67

Activation of Bacterial Translation by Small RNA Molecules

The ribosome-binding Shine−Dalgarno 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.

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Inhibition of Bacterial Translation by Small RNA Molecules

The ribosome-binding Shine−Dalgarno 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.

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Cis Regulation by Riboswitches

Riboswitch = domain of an mRNA that can bind a small-molecule ligandThe 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.

Slide70

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.

Slide71

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 SalmonellaIn one orientation fljB is expressed with a repressor for fljC gene.In another orientation, only fljC is expressed.The process is called phase variation.

Slide72

Phase Variation – Regulation of Flagellin Genes

Slide73

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

μ

)

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

conversion

a

Alternative expression of two mating types of yeast, a and

α

, creates cells of different mating types that can mate and undergo meiosis.

Antigenic variation (

trypanosomes)

b

Varies

Nonreciprocal gene

conversion

a

Successive expression of different genes encoding the variable surface glycoproteins (

VSGs

) allows evasion of host immune response.

a

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

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Features of Eukaryotic Gene Regulation

Access of eukaryotic promoters to RNA polymerase is hindered by chromatin structure.thus requires remodeling chromatinPositive regulation mechanisms predominate and are required for even a basal level of gene expression.Eukaryotic gene expression requires a complicated set of proteins.

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Three Features of Transcriptionally Active Chromatin

Euchromatin = less-condensed chromatin, distinguished from transcriptionally inactive heterochromatinChromatin remodeling of transcriptionally active genes:nucleosomes repositioned histone variantscovalent modifications to nucleosomes

Slide76

Nucleosomes Can Be Restructured by Specific Protein Complexes

SWI/SNF (SWItch/Sucrose NonFermentable) complexremodels chromatin to irregularly space nucleosomesstimulates binding of transcription factorsworks with proteins of ISWI (imitation switch) familyATP-dependent alteration of spacing between nucleosomes, and so on

Slide77

Nucleosome Remolding

Eukaryotic cells generally have 9−10 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 repairSWR1 – promotes subunit exchange in nucleosomes inducing histone variants Ie: H2Az – found in transcriptionally active regions

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Covalent Modification of Histones

MethylationPhosphorylationAcetylationUbiquitinationSumoylationOccur mostly in the N-terminal domain of the histones found near the exterior of the nucleosome particle

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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 activationrecruits histone acetyltransferases (HATs) that then acetylate a particular Lysreversed by histone deacetylases (HDACs) that make chromatin inactiveAcetylation of Lys results in decreased affinity of histone for DNA.

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TABLE 28-2

Some Enzyme Complexes That Catalyze Chromatin Structural Changes Associated with Transcription

Enzyme

complex

a

Oligomeric

structure (number of polypeptides)

Source

Activities

Histone movement, replacement, or editing, requiring ATP

SWI/SNF family

8–17

M

r

> 106

Eukaryotes

Nucleosome remodeling; transcriptional activation

ISWI family

2–4

Eukaryotes

Nucleosome remodeling; transcriptional repression; transcriptional activation in some cases

CHD family

1–10

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

Histone modification

GCN5-ADA2-ADA3

3

Yeast

GCN5 has type A HAT activity

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

Histone chaperones not requiring ATP

HIRA

1

Eukaryotes

Eukaryotes Deposition of H3.3 during transcription

a

The

abbreviations for eukaryotic genes and proteins are often more confusing or obscure than those used for bacteria. SWI (

swi

tching) 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

i

mitation

SWI

. CHD is

c

hromodomain

,

h

elicase

,

D

NA binding; INO80,

in

ositol

-requiring

80

; and SWR1,

SW

i2/Snf2-

r

elated 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

nu

cleosome

a

cetyltransferase

of H

4

; ESA1, essential SAS2-related

acetyltransferase

. HIRA is

his

tone regulator

A

.

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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.

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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.

Slide83

RNA Polymerase II Requires Five Types of Proteins

Transcription activators (enhancers)proteins that bind to upstream activator sequences (UASs)Architectural regulators to facilitate DNA loopingChromatin modification/remodeling proteinsCoactivatorsact indirectly (with other proteins, not with DNA)Basal (general) transcription factors

Slide84

Enhancer Proteins Are Diverse

Can bind thousands of nucleotides away from the TATA box of the promoterCan have DNA-binding, protein-binding, and/or signal molecule-binding domainscan bind with multiple proteinsSome regulate a few genes; some regulate many hundreds of genes

Slide85

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

Slide86

Coactivators Assist RNA Polymerase

Mediator complex binds to carboxyl-terminal domain(CTD) of RNA Pol IIrequired for both basal and regulated transcription at many promoterslater provides assembly surface for other complexes TATA-binding protein is first component of preinitiation complex (PIC) at the typical TATA box of a promoter

Slide87

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.

Slide88

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)

Slide89

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.

Slide90

TABLE 28-3

Genes of

Galactose

Metabolism in Yeast

Gene

Protein function

Chromosomal location

Protein size (number of residues)

Relative protein expression in different carbon sources

Glucose

Glycerol

Galactose

Regulated genes

GAL1

Galactokinase

II

528

–

–

+++

GAL2

Galactose

permease

XII

574

–

–

+++

PGM2

Phosphoglucomutase

XIII

569

+

+

++

GAL7

Galactose

1-phosphate

uridylyltransferase

II

365

–

–

+++

GAL10

UDP-glucose 4-epimerase

II

699

–

–

+++

MEL1

α

-Galactosidase

II

453

–

+

++

Regulatory Genes

GAL3

Inducer

IV

520

–

+

++

GAL4

Transcriptional activator

XVI

881

+/–

+

+

GAL80

Transcriptional inhibitor

XIII

435

+

+

++

Source: Information from R. Reece and A. Platt,

Bioessays

19:1001, 1997.

Slide91

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.

Slide92

Two Types of Hormone Receptors

Monomeric Type 1 (NR)―receptors for sex hormones and glucocorticoidsfound in cytoplasm in complex with Hsp70when hormone binds, Hsp70 dissociatesreceptor dimerizesexposes nuclear localization regionso hormone-receptor complex travels to nucleus to be transcriptional activator

Slide93

Type II (includes thyroid hormone receptor; TR)found in nucleus bound to DNA and corepressor retinoid X receptor (RXR)hormone binds, corepressor dissociatesreceptor-hormone complex then activates transcription

Two Types of Hormone Receptors

Slide94

Eukaryotic Gene Expression Is Also Regulated by Peptide Hormones

2 messengers lead to activation of transcription factorsExample: -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.

Slide95

Four Mechanisms of Translation Regulation

Phosphorylation of translation initiation factorsTranslational repressors (typically bind to 3’ UTR)Disruption of eIF4E and eIF4G interactionsRNA-mediated regulation (gene silencing)

Slide96

Micro-RNAs Prevent Translation of mRNA

Micro-RNAs (miRNAs) silence genes by binding to mRNAscan prevent transcription of the mRNA by cleaving it (via endonucleases Drosha or Dicer) or by blocking itSome miRNAs are made briefly during development; they are called small temporal RNAs (stRNAs).

Slide97

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.

Slide98

Gene Silencing by RNA Interference

Slide99

ncRNAs Also Regulate Gene Expression

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

Slide100

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

Slide101

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.

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Homeotic versus Maternal Genes

Homeotic genes – specify development of organs and appendages in a particular body segmentHox genes – homeobox – contains a conserved sequence present in all of these genesMaternal genes – genes expressed in nurse and follicle cells that affect initial polarity and axes development (bicoid and nanos)

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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 poleLack of Bicoid results in an embryo without a head or thorax and two abdomens.Nanos – translational repressor protein deposited at posterior of the egg

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Translational Regulation of Anterior-Posterior Development

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Homeotic Genes

Often found in clusters and are highly conserved amount animals

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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.

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Developmental Potential of Stem Cells

Embryonic stem cells pluripotent cells of the blastocyst used in embryonic stem cell researchAdult stem cellslimited potential compared with embryonic hematopoietic stem cells – can give rise to different cells in niche or microenvironmentconsidered multipotentexample: 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

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Developmental Potential of Stem Cells

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Chapter 28: Summary

positive and negative gene regulation occurs in both prokaryotic and eukaryotic organismsthere are many useful examples of gene regulation – lac operon, trp operon, and so onregulator proteins contain domains that interact with DNA, RNA, small molecules, and so onncRNAs are involved in regulation of gene expressioneukaryotic gene regulation is complex and involves chromatin remodeling, combinatory control, intercellular and intracellular signals, and gene silencingdevelopment in animals is controlled by cascades of regulatory proteins and their precise position within the embryo

In this chapter, we learned that: