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3: Prokaryotic Regulation of Genetic Expression - Biology

3:  Prokaryotic Regulation of Genetic Expression - Biology


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Prokaryotic Regulation of Genetic Expression

Regulation of transcription: Operons and operators

Vocabulary

structural genes: genes which encode information for a protein

“gene expression”= transcription (+ translation when describing structural genes)

for our class discussion, we will presume that when a structural gene is transcribed, the mRNA will be translated into protein thus “gene expression” results in protein production

-ase= common ending for enzymes

lactose: also called “milk sugar”, lactose is a disaccharide of glucose and galactose joined by a glycosidic bond.

lactose= galactose-glucose

promoter: special DNA sequence to which RNA polymerase binds to start transcription

operon: DNA sequence encoding promoter and operator sites and structural genes they control

operator: special DNA sequence to which repressor proteins may bind to block transcription by RNA polymerase; the “on-off” switch for transcription

allosteric proteins: proteins which can have 2 different forms

lac repressor protein: an allosteric protein which has 2 forms:

an active form in which the repressor bind to the lac operator and blocks transcription of the lac operon structural genes when lactose is absent and …an inactive form which cannot bind the lac operator. The inactive form is dominant when lactose is present and the inducer allolactose binds to the lac repressor protein.

Intro survival notes:

Gene expression and protein synthesis are costly to cells (requires lots of “building blocks” and energy.

(From Bauman) ...most bacterial genes are expressed constantly= constitutive expression. These genes include genes for tRNAs, rRNAs and structural genes for proteins constantly needed for example, ribosomal proteins and enzymes used in glycolysis.

However some gene products may be required only under certain conditions. For example, the gene encoding the enzyme beta-galactosidase need only be expressed if lactose is present in the environment. Beta-galactosidase is the bacterial equivalent of human “lactase”. It catalyzes hydrolysis of the glycosidic bond between the residues of glucose and galactose in milk sugar, lactose:

Lactose-> beta-galactosidase -> glucose + galactose

Bacteria which can turn on and turn off transcription of genes like beta-galactosidase will have a survival advantage over other bacteria which cannot regulate gene expression (Why?)

Regulation of Bacterial Gene Expression: transcription control

1. Three types of gene expression

  • constitutive: continual transcription (and continual synthesis of encoded proteins)
  • inducible: made only when substrate or signal molecule is present e.g., enzymes for lactose transport/metabolism in E. coli
  • repressible: produced only when signal molecule is scarce

2. Operon: group of genes whose transcription is coordinately turned on or off; under control of operator and promoter

3. Operator: specific DNA sequence which lies between promoter and 1st codon of gene. Repressor proteins bind operator and

block ability of RNA polymerase to bind promoter/ transcribe “downstream” structural genes. Repressor proteins are allosteric proteins. The have one binding site for a DNA operator sequence and a second binding site for an “inducer” molecule for e.g. allolactose in the lac operon ( or a “corepressor” molecule e.g. trp operon )

Inducible operons. Gene transcription turned only when substrate/signal is present. Usually genes for catabolic enzymes example: E. coli lac operon. Jacob and Monod 1961

1. Lac operon consists of 3 structural genes (lacZ, Y and A) , promoter and operator.

Lac operon is inducible.

Structural gene gene product (enzyme/transport protein)

lacZ beta-galactosidase

lacY lactose transport protein/ galactoside permease

lacA galactoside transacetylase

-----------------------------------------------------------------------------------------------------

2. Regulatory/repressor gene, lacI

LacI is the lac repressor protein.

The lac repressor protein is a DNA binding protein, which when active can bind to the lac operator, blocking transcription/expression of lacZ, Y and A in absence of lactose (survival note: it would be wasteful for the bacterium to make these proteins if lactose is not present). Lac repressor gene has its own promoter and is constitutively expressed. The lac repressor gene is NOT part of the lac operon

3. lac operon when NO LACTOSE AVAILABLE (fill-in cartoon below, double dotted lines represent a single strand of DNA)

DNA ___________________________________________________________

------------------------------------------------------------------------------------------------

lac I Promoterlac Operatorlac lacZ lacY lacA

How should your cartoon look?

lac repressor protein binds lac operator in absence of lactose, blocks transcription of lac genes by RNA polymerase. Note: all repression is “leaky”, that is repressor binds and releases operator in a concentration dependent manner. When lactose is absent, most repressor proteins are in the active form, blocking most (but not all) transcription of the lac structural genes)

4. ADD LACTOSE: (How does your cartoon differ from above?)lactose-> inducer allolactose binds to allosteric site on lac repressor, causes it to change shape so it can no longer bind operator.

DNA ___________________________________________________________

------------------------------------------------------------------------------------------------

lac I Promoterlac Operatorlac lacZ lacY lacA

DNA ___________________________________________________________

------------------------------------------------------------------------------------------------

lac I Promoterlac Operatorlac lacZ lacY lacA

5. Now RNA polymerase can start transcribing the lac genes and cell can makes transport protein and beta-galactosidase, cell starts transporting and breaking down lactose at high rate

6. When lactose used up, allolactose levels drop/release lac repressor, repressor regains shape, binds operator, turns off lac gene transcription

DNA ___________________________________________________________

------------------------------------------------------------------------------------------------

lac I Promoterlac Operatorlac lacZ lacY lacA

What would happen if…..

What if….

E.coli had a mutation so that the lac repressor could not bind DNA?

“ “ had a mutation so that the mutant lac repressor protein could NOT bind allolactose, the inducer?

“” had a mutation so that the operator could not bind normal lac repressor protein?

Deleted section from previous semesters:

I. Regulation of metabolism: 2 ways

A. Change activity of enzymes: allosteric sites/allosteric enzymes

1.binding sites other than active site=allosteric sites, bind “effectors”

2. binding of effector changes 3-D shape of enzyme

3. Two kinds of effectors:

a. allosteric activators: bind allosteric site, change enzyme so that it is MORE ACTIVE/turned on/activate enzyme

b. allosteric inhibitors: bind allosteric site, change enzyme shape so it is less active/turned off /inhibit enzyme e.g.,: end product inhibition

4. Provides rapid response to changes in substrates, need for endproducts

B. Change expression of genes that is regulate transcription (or even translation)


Contents

Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively utilised point is Transcription Initiation:

In eukaryotes, the accessibility of large regions of DNA can depend on its chromatin structure, which can be altered as a result of histone modifications directed by DNA methylation, ncRNA, or DNA-binding protein. Hence these modifications may up or down regulate the expression of a gene. Some of these modifications that regulate gene expression are inheritable and are referred to as epigenetic regulation.

Structural Edit

Transcription of DNA is dictated by its structure. In general, the density of its packing is indicative of the frequency of transcription. Octameric protein complexes called histones together with a segment of DNA wound around the eight histone proteins (together referred to as a nucleosome) are responsible for the amount of supercoiling of DNA, and these complexes can be temporarily modified by processes such as phosphorylation or more permanently modified by processes such as methylation. Such modifications are considered to be responsible for more or less permanent changes in gene expression levels. [2]

Chemical Edit

Methylation of DNA is a common method of gene silencing. DNA is typically methylated by methyltransferase enzymes on cytosine nucleotides in a CpG dinucleotide sequence (also called "CpG islands" when densely clustered). Analysis of the pattern of methylation in a given region of DNA (which can be a promoter) can be achieved through a method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment, whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA sequencing or by methods developed to quantify SNPs, such as Pyrosequencing (Biotage) or MassArray (Sequenom), measuring the relative amounts of C/T at the CG dinucleotide. Abnormal methylation patterns are thought to be involved in oncogenesis. [3]

Histone acetylation is also an important process in transcription. Histone acetyltransferase enzymes (HATs) such as CREB-binding protein also dissociate the DNA from the histone complex, allowing transcription to proceed. Often, DNA methylation and histone deacetylation work together in gene silencing. The combination of the two seems to be a signal for DNA to be packed more densely, lowering gene expression. [ citation needed ]

Regulation of transcription thus controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymerase can be regulated by several mechanisms. Specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e., sigma factors used in prokaryotic transcription). Repressors bind to the Operator, coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene. The image to the right demonstrates regulation by a repressor in the lac operon. General transcription factors position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA. Activators enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA. Enhancers are sites on the DNA helix that are bound by activators in order to loop the DNA bringing a specific promoter to the initiation complex. Enhancers are much more common in eukaryotes than prokaryotes, where only a few examples exist (to date). [4] Silencers are regions of DNA sequences that, when bound by particular transcription factors, can silence expression of the gene.

In vertebrates, the majority of gene promoters contain a CpG island with numerous CpG sites. [5] When many of a gene's promoter CpG sites are methylated the gene becomes silenced. [6] Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. [7] However, transcriptional silencing may be of more importance than mutation in causing progression to cancer. For example, in colorectal cancers about 600 to 800 genes are transcriptionally silenced by CpG island methylation (see regulation of transcription in cancer). Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs. [8] In breast cancer, transcriptional repression of BRCA1 may occur more frequently by over-expressed microRNA-182 than by hypermethylation of the BRCA1 promoter (see Low expression of BRCA1 in breast and ovarian cancers).

One of the cardinal features of addiction is its persistence. The persistent behavioral changes appear to be due to long-lasting changes, resulting from epigenetic alterations affecting gene expression, within particular regions of the brain. [9] Drugs of abuse cause three types of epigenetic alteration in the brain. These are (1) histone acetylations and histone methylations, (2) DNA methylation at CpG sites, and (3) epigenetic downregulation or upregulation of microRNAs. [9] [10] (See Epigenetics of cocaine addiction for some details.)

Chronic nicotine intake in mice alters brain cell epigenetic control of gene expression through acetylation of histones. This increases expression in the brain of the protein FosB, important in addiction. [11] Cigarette addiction was also studied in about 16,000 humans, including never smokers, current smokers, and those who had quit smoking for up to 30 years. [12] In blood cells, more than 18,000 CpG sites (of the roughly 450,000 analyzed CpG sites in the genome) had frequently altered methylation among current smokers. These CpG sites occurred in over 7,000 genes, or roughly a third of known human genes. The majority of the differentially methylated CpG sites returned to the level of never-smokers within five years of smoking cessation. However, 2,568 CpGs among 942 genes remained differentially methylated in former versus never smokers. Such remaining epigenetic changes can be viewed as “molecular scars” [10] that may affect gene expression.

In rodent models, drugs of abuse, including cocaine, [13] methampheamine, [14] [15] alcohol [16] and tobacco smoke products, [17] all cause DNA damage in the brain. During repair of DNA damages some individual repair events can alter the methylation of DNA and/or the acetylations or methylations of histones at the sites of damage, and thus can contribute to leaving an epigenetic scar on chromatin. [18]

Such epigenetic scars likely contribute to the persistent epigenetic changes found in addiction.

In mammals, methylation of cytosine (see Figure) in DNA is a major regulatory mediator. Methylated cytosines primarily occur in dinucleotide sequences where cytosine is followed by a guanine, a CpG site. The total number of CpG sites in the human genome is approximately 28 million. [19] and generally about 70% of all CpG sites have a methylated cytosine. [20]

In a rat, a painful learning experience, contextual fear conditioning, can result in a life-long fearful memory after a single training event. [21] Cytosine methylation is altered in the promoter regions of about 9.17% of all genes in the hippocampus neuron DNA of a rat that has been subjected to a brief fear conditioning experience. [22] The hippocampus is where new memories are initially stored.

Methylation of CpGs in a promoter region of a gene represses transcription [23] while methylation of CpGs in the body of a gene increases expression. [24] TET enzymes play a central role in demethylation of methylated cytosines. Demethylation of CpGs in a gene promoter by TET enzyme activity increases transcription of the gene. [25]

When contextual fear conditioning is applied to a rat, more than 5,000 differentially methylated regions (DMRs) (of 500 nucleotides each) occur in the rat hippocampus neural genome both one hour and 24 hours after the conditioning in the hippocampus. [22] This causes about 500 genes to be up-regulated (often due to demethylation of CpG sites in a promoter region) and about 1,000 genes to be down-regulated (often due to newly formed 5-methylcytosine at CpG sites in a promoter region). The pattern of induced and repressed genes within neurons appears to provide a molecular basis for forming the first transient memory of this training event in the hippocampus of the rat brain. [22]

After the DNA is transcribed and mRNA is formed, there must be some sort of regulation on how much the mRNA is translated into proteins. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript. These processes occur in eukaryotes but not in prokaryotes. This modulation is a result of a protein or transcript that, in turn, is regulated and may have an affinity for certain sequences.

Three prime untranslated regions (3'-UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally influence gene expression. [26] Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.

The 3'-UTR often contains miRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.

As of 2014, the miRBase web site, [27] an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes). [28] Freidman et al. [28] estimate that >45,000 miRNA target sites within human mRNA 3'-UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. [29] Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold). [30] [31]

The effects of miRNA dysregulation of gene expression seem to be important in cancer. [32] For instance, in gastrointestinal cancers, a 2015 paper identified nine miRNAs as epigenetically altered and effective in down-regulating DNA repair enzymes. [33]

The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depressive disorder, Parkinson's disease, Alzheimer's disease and autism spectrum disorders. [34] [35] [36]

The translation of mRNA can also be controlled by a number of mechanisms, mostly at the level of initiation. Recruitment of the small ribosomal subunit can indeed be modulated by mRNA secondary structure, antisense RNA binding, or protein binding. In both prokaryotes and eukaryotes, a large number of RNA binding proteins exist, which often are directed to their target sequence by the secondary structure of the transcript, which may change depending on certain conditions, such as temperature or presence of a ligand (aptamer). Some transcripts act as ribozymes and self-regulate their expression.

    is a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the expression of an enzyme.
  • The induction of heat shock proteins in the fruit fly Drosophila melanogaster.
  • The Lac operon is an interesting example of how gene expression can be regulated.
  • Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV).
  • Gal4 is a transcriptional activator that controls the expression of GAL1, GAL7, and GAL10 (all of which code for the metabolic of galactose in yeast). The GAL4/UAS system has been used in a variety of organisms across various phyla to study gene expression. [37]

Developmental biology Edit

A large number of studied regulatory systems come from developmental biology. Examples include:

  • The colinearity of the Hox gene cluster with their nested antero-posterior patterning
  • Pattern generation of the hand (digits - interdigits): the gradient of sonic hedgehog (secreted inducing factor) from the zone of polarizing activity in the limb, which creates a gradient of active Gli3, which activates Gremlin, which inhibits BMPs also secreted in the limb, results in the formation of an alternating pattern of activity as a result of this reaction-diffusion system.
  • Somitogenesis is the creation of segments (somites) from a uniform tissue (Pre-somitic Mesoderm). They are formed sequentially from anterior to posterior. This is achieved in amniotes possibly by means of two opposing gradients, Retinoic acid in the anterior (wavefront) and Wnt and Fgf in the posterior, coupled to an oscillating pattern (segmentation clock) composed of FGF + Notch and Wnt in antiphase. [38]
  • Sex determination in the soma of a Drosophila requires the sensing of the ratio of autosomal genes to sex chromosome-encoded genes, which results in the production of sexless splicing factor in females, resulting in the female isoform of doublesex. [39]

Up-regulation and down-regulation Edit

Up-regulation is a process that occurs within a cell triggered by a signal (originating internal or external to the cell), which results in increased expression of one or more genes and as a result the protein(s) encoded by those genes. Conversely, down-regulation is a process resulting in decreased gene and corresponding protein expression.

    occurs, for example, when a cell is deficient in some kind of receptor. In this case, more receptor protein is synthesized and transported to the membrane of the cell and, thus, the sensitivity of the cell is brought back to normal, reestablishing homeostasis. occurs, for example, when a cell is overstimulated by a neurotransmitter, hormone, or drug for a prolonged period of time, and the expression of the receptor protein is decreased in order to protect the cell (see also tachyphylaxis).

Inducible vs. repressible systems Edit

Gene Regulation can be summarized by the response of the respective system:

  • Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
  • Repressible systems - A repressible system is on except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to "repress expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.

The GAL4/UAS system is an example of both an inducible and repressible system. Gal4 binds an upstream activation sequence (UAS) to activate the transcription of the GAL1/GAL7/GAL10 cassette. On the other hand, a MIG1 response to the presence of glucose can inhibit GAL4 and therefore stop the expression of the GAL1/GAL7/GAL10 cassette. [40]

Theoretical circuits Edit

  • Repressor/Inducer: an activation of a sensor results in the change of expression of a gene
  • negative feedback: the gene product downregulates its own production directly or indirectly, which can result in
    • keeping transcript levels constant/proportional to a factor
    • inhibition of run-away reactions when coupled with a positive feedback loop
    • creating an oscillator by taking advantage in the time delay of transcription and translation, given that the mRNA and protein half-life is shorter
    • signal amplification
    • bistable switches when two genes inhibit each other and both have positive feedback
    • pattern generation

    In general, most experiments investigating differential expression used whole cell extracts of RNA, called steady-state levels, to determine which genes changed and by how much. These are, however, not informative of where the regulation has occurred and may mask conflicting regulatory processes (see post-transcriptional regulation), but it is still the most commonly analysed (quantitative PCR and DNA microarray).

    When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:


    Biology 171


    Each somatic cell in the body generally contains the same DNA. A few exceptions include red blood cells, which contain no DNA in their mature state, and some immune system cells that rearrange their DNA while producing antibodies. In general, however, the genes that determine whether you have green eyes, brown hair, and how fast you metabolize food are the same in the cells in your eyes and your liver, even though these organs function quite differently. If each cell has the same DNA, how is it that cells or organs are different? Why do cells in the eye differ so dramatically from cells in the liver?

    Whereas each cell shares the same genome and DNA sequence, each cell does not turn on, or express, the same set of genes. Each cell type needs a different set of proteins to perform its function. Therefore, only a small subset of proteins is expressed in a cell. For the proteins to be expressed, the DNA must be transcribed into RNA and the RNA must be translated into protein. In a given cell type, not all genes encoded in the DNA are transcribed into RNA or translated into protein because specific cells in our body have specific functions. Specialized proteins that make up the eye (iris, lens, and cornea) are only expressed in the eye, whereas the specialized proteins in the heart (pacemaker cells, heart muscle, and valves) are only expressed in the heart. At any given time, only a subset of all of the genes encoded by our DNA are expressed and translated into proteins. The expression of specific genes is a highly regulated process with many levels and stages of control. This complexity ensures the proper expression in the proper cell at the proper time.

    Learning Objectives

    By the end of this section, you will be able to do the following:

    • Discuss why every cell does not express all of its genes all of the time
    • Describe how prokaryotic gene regulation occurs at the transcriptional level
    • Discuss how eukaryotic gene regulation occurs at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels

    For a cell to function properly, necessary proteins must be synthesized at the proper time and place. All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression . Whether in a simple unicellular organism or a complex multi-cellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be internal chemical mechanisms that control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.

    The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the time.

    The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.

    Prokaryotic versus Eukaryotic Gene Expression

    To understand how gene expression is regulated, we must first understand how a gene codes for a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different manners.

    Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesize a protein, the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription. All of the subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level.

    Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In eukaryotic cells, the DNA is contained inside the cell’s nucleus and there it is transcribed into RNA. The newly synthesized RNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the RNA into protein. The processes of transcription and translation are physically separated by the nuclear membrane transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. The regulation of gene expression can occur at all stages of the process ((Figure)). Regulation may occur when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors ( epigenetic level), when the RNA is transcribed ( transcriptional level), when the RNA is processed and exported to the cytoplasm after it is transcribed ( post-transcriptional level), when the RNA is translated into protein ( translational level), or after the protein has been made ( post-translational level).


    The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized in (Figure). The regulation of gene expression is discussed in detail in subsequent modules.

    Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms
    Prokaryotic organisms Eukaryotic organisms
    Lack a membrane-bound nucleus Contain nucleus
    DNA is found in the cytoplasm DNA is confined to the nuclear compartment
    RNA transcription and protein formation occur almost simultaneously RNA transcription occurs prior to protein formation, and it takes place in the nucleus. Translation of RNA to protein occurs in the cytoplasm.
    Gene expression is regulated primarily at the transcriptional level Gene expression is regulated at many levels (epigenetic, transcriptional, nuclear shuttling, post-transcriptional, translational, and post-translational)

    Prokaryotic cells can only regulate gene expression by controlling the amount of transcription. As eukaryotic cells evolved, the complexity of the control of gene expression increased. For example, with the evolution of eukaryotic cells came compartmentalization of important cellular components and cellular processes. A nuclear region that contains the DNA was formed. Transcription and translation were physically separated into two different cellular compartments. It therefore became possible to control gene expression by regulating transcription in the nucleus, and also by controlling the RNA levels and protein translation present outside the nucleus.

    Most gene regulation is done to conserve cell resources. However, other regulatory processes may be defensive. Cellular processes such as developed to protect the cell from viral or parasitic infections. If the cell could quickly shut off gene expression for a short period of time, it would be able to survive an infection when other organisms could not. Therefore, the organism evolved a new process that helped it survive, and it was able to pass this new development to offspring.

    Section Summary

    While all somatic cells within an organism contain the same DNA, not all cells within that organism express the same proteins. Prokaryotic organisms express most of their genes most of the time. However, some genes are expressed only when they are needed. Eukaryotic organisms, on the other hand, express only a subset of their genes in any given cell. To express a protein, the DNA is first transcribed into RNA, which is then translated into proteins, which are then targeted to specific cellular locations. In prokaryotic cells, transcription and translation occur almost simultaneously. In eukaryotic cells, transcription occurs in the nucleus and is separate from the translation that occurs in the cytoplasm. Gene expression in prokaryotes is mostly regulated at the transcriptional level (some epigenetic and post-translational regulation is also present), whereas in eukaryotic cells, gene expression is regulated at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels.

    Free Response

    Name two differences between prokaryotic and eukaryotic cells and how these differences benefit multicellular organisms.

    Eukaryotic cells have a nucleus, whereas prokaryotic cells do not. In eukaryotic cells, DNA is confined within the nuclear region. Because of this, transcription and translation are physically separated. This creates a more complex mechanism for the control of gene expression that benefits multicellular organisms because it compartmentalizes gene regulation.

    Gene expression occurs at many stages in eukaryotic cells, whereas in prokaryotic cells, control of gene expression only occurs at the transcriptional level. This allows for greater control of gene expression in eukaryotes and more complex systems to be developed. Because of this, different cell types can arise in an individual organism.

    Describe how controlling gene expression will alter the overall protein levels in the cell.

    The cell controls which proteins are expressed and to what level each protein is expressed in the cell. Prokaryotic cells alter the transcription rate to turn genes on or off. This method will increase or decrease protein levels in response to what is needed by the cell. Eukaryotic cells change the accessibility (epigenetic), transcription, or translation of a gene. This will alter the amount of RNA and the lifespan of the RNA to alter the amount of protein that exists. Eukaryotic cells also control protein translation to increase or decrease the overall levels. Eukaryotic organisms are much more complex and can manipulate protein levels by changing many stages in the process.

    Glossary


    Expression of Genes

    Gene regulation makes cells different

    Gene regulation is how a cell controls which genes, out of the many genes in its genome, are “turned on” (expressed). Thanks to gene regulation, each cell type in your body has a different set of active genes—despite the fact that almost all the cells of your body contain the exact same DNA. These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job. Ultimately gene expression can involve changes in transcription or translation, but in eukaryotes, most gene expression control occurs at transcription.

    For example, one of the jobs of the liver is to remove toxic substances like alcohol from the bloodstream. To do this, liver cells express genes encoding subunits (pieces) of an enzyme called alcohol dehydrogenase. This enzyme breaks alcohol down into a non-toxic molecule. The neurons in a person’s brain don’t remove toxins from the body, so they keep these genes unexpressed, or “turned off.” Similarly, the cells of the liver don’t send signals using neurotransmitters, so they keep neurotransmitter genes turned off (Figure 1).

    Figure 1. Different cells have different genes “turned on.”

    There are many other genes that are expressed differently between liver cells and neurons (or any two cell types in a multicellular organism like yourself).

    How do cells “decide” which genes to turn on?

    Now there’s a tricky question! Different cell types express different sets of genes, as we saw above. However, two different cells of the same type may also have different gene expression patterns depending on their environment and internal state.

    Broadly speaking, we can say that a cell’s gene expression pattern is determined by information from both inside and outside the cell.

    • Examples of information from inside the cell: the proteins it inherited from its mother cell, whether its DNA is damaged, and how much ATP it has.
    • Examples of information from outside the cell: chemical signals from other cells, mechanical signals from the extracellular matrix, and nutrient levels.

    How do these cues help a cell “decide” what genes to express? Cells don’t make decisions in the sense that you or I would. Instead, they have molecular pathways that convert information—such as the binding of a chemical signal to its receptor—into a change in gene expression.

    As an example, let’s consider how cells respond to growth factors. A growth factor is a chemical signal from a neighboring cell that instructs a target cell to grow and divide. We could say that the cell “notices” the growth factor and “decides” to divide, but how do these processes actually occur?

    Figure 2. Growth factor prompting cell division

    • The cell detects the growth factor through physical binding of the growth factor to a receptor protein on the cell surface.
    • Binding of the growth factor causes the receptor to change shape, triggering a series of chemical events in the cell that activate proteins called transcription factors.
    • The transcription factors bind to certain sequences of DNA in the nucleus and cause transcription of cell division-related genes.
    • The products of these genes are various types of proteins that make the cell divide (drive cell growth and/or push the cell forward in the cell cycle).

    This is just one example of how a cell can convert a source of information into a change in gene expression. There are many others, and understanding the logic of gene regulation is an area of ongoing research in biology today.

    Growth factor signaling is complex and involves the activation of a variety of targets, including both transcription factors and non-transcription factor proteins.

    In Summary: Expression of Genes

    • Gene regulation is the process of controlling which genes in a cell’s DNA are expressed (used to make a functional product such as a protein).
    • Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA.
    • The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, giving it its unique properties.
    • In eukaryotes like humans, gene expression involves many steps, and gene regulation can occur at any of these steps. However, many genes are regulated primarily at the level of transcription.

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    Toehold switches: de-novo-designed regulators of gene expression

    Efforts to construct synthetic networks in living cells have been hindered by the limited number of regulatory components that provide wide dynamic range and low crosstalk. Here, we report a class of de-novo-designed prokaryotic riboregulators called toehold switches that activate gene expression in response to cognate RNAs with arbitrary sequences. Toehold switches provide a high level of orthogonality and can be forward engineered to provide average dynamic range above 400. We show that switches can be integrated into the genome to regulate endogenous genes and use them as sensors that respond to endogenous RNAs. We exploit the orthogonality of toehold switches to regulate 12 genes independently and to construct a genetic circuit that evaluates 4-input AND logic. Toehold switches, with their wide dynamic range, orthogonality, and programmability, represent a versatile and powerful platform for regulation of translation, offering diverse applications in molecular biology, synthetic biology, and biotechnology.

    Figures

    Figure 1. Toehold switch design and in…

    Figure 1. Toehold switch design and in vivo characterization

    Figure 2. Assessment of toehold switch orthogonality

    Figure 2. Assessment of toehold switch orthogonality

    Figure 3. Forward engineering and thermodynamic analysis…

    Figure 3. Forward engineering and thermodynamic analysis of toehold switches

    Figure 4. Toehold switch activated by mRNA…

    Figure 4. Toehold switch activated by mRNA and endogenous small RNA triggers

    Figure 5. Synthetic regulation of endogenous genes

    Figure 5. Synthetic regulation of endogenous genes

    Figure 6. Simultaneous regulation of gene expression…

    Figure 6. Simultaneous regulation of gene expression by twelve toehold switches

    3.4-kb polycistronic mRNAs used for multiplexing studies. Each reporter has its own switch RNA that can be independently activated by its cognate trigger RNA. (B) Percentage of cells expressing each of the four reporters for a set of 24 different trigger RNA combinations. Gray and colored circles are used to identify the particular trigger RNA being expressed and the corresponding switch RNA. See also Figure S5, Table S6.


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