Explain the Importance of Gene Regulation in Both Prokaryotes And

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Unit 4 - Gene Expression and Module 4A – Control of Gene Expression Genetic Technology Every cell contains thousands of genes 4A. Control of Gene Expression which code for proteins. However, every gene is not actively 4B. Biotechnology producing proteins at all times. 4C. Genomics In this module, we will examine some 4D. Genes and Development of the factors that help regulate when a gene is active, and how strongly it is expressed. 1 2

Objective # 1 Objective 1 Prokaryotes: Explain the importance of gene ¾ are unicellular or colonial regulation in both prokaryotes ¾ evolved to quickly exploit transient and eukaryotes. resources ¾ main role of gene regulation is to allow cells to adjust to changing conditions ¾ in the same cell, different genes are active at different times 3 4

Objective 1 Objective # 2 Eukaryotes: ¾ mostly complex multicellular organisms List and describe the different ¾ evolved the ability to maintain a stable levels of gene regulation, and internal environment (homeostasis) identify the level where genes ¾ main role of gene regulation is to allow are most commonly regulated. specialization and division of labor among cells ¾ at the same time, different genes are active in different cells 5 6

1 Objective 2 Objective 2

We can classify levels of gene To be expressed, a gene must be regulation into 2 main categories: transcribed into m-RNA, the m-RNA must be translated into a protein, and ¾ Transcriptional controls - factors that the protein must become active. regulate transcription ¾ Posttranscriptional controls – factors Gene regulation can theoretically occur that regulate any step in gene at any step in this process. expression after transcription is complete

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Levels of 4. Destruction of 5.Translation. Many gene control the transcript. Many enzymes proteins take part in in degrade mRNA, and translation, and gene expression can regulating the activity Eukaryotes be regulated by PO Amino modulating the 4 or availability of any of acid DNA degree to which the transcript is them can alter the rate protected. of gene expression. Nuclear 3′ membrane PO4 3′ RNA Completed polymerase 5′ Cap polypeptide tRNA 1. Initiation of 5′ transcription. Small Most control of gene 3′ ribosomal 5′ expression is achieved 5′ Primary subunit by regulating the RNA transcript Nuclear frequency of pore Ribosome moves transcription initiation. 5′ toward 3′ end 3′ Large Exons Cap ribosomal 3′ Poly-A mRNA subunit tail Poly-A Cytoplasm Introns tail Ribosome mRNA 3′ 3. Passage through the 2. RNA splicing. Gene nuclear membrane. 6. Post-translational modification. Phosphorylation or expression can be controlled Gene expression can be other chemical modifications can alter the activity of by altering the rate of splicing regulated by controlling access in eukaryotes. to or efficiency of transport a protein after it is produced. channels. 9 10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Objective 2 Objective # 3

It is most efficient to regulate genes at the first step of gene expression – Explain what regulatory namely transcription. proteins do and describe how Both prokaryotes and eukaryotes rely they identify specific sequences primarily on transcriptional controls to regulate gene expression. on the DNA double helix.

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2 Objective 3 Objective 3

In order to regulate transcription, Biologists used to think the DNA special proteins called regulatory double helix had to unwind and proteins must bind to specific regions separate before proteins could “read” of the DNA called regulatory the sequence of base pairs. sequences. However, careful study has revealed 2 How does each regulatory protein helical grooves winding around the recognize the appropriate regulatory DNA molecule. The deeper one is sequence on the DNA? called the major groove and the shallower one is the minor groove. 13 14

Objective 3 The Helix-Turn-Helix Motif

Regulatory proteins can read DNA α Helix Turn sequences by inserting bent regions (Recognition helix) of the protein chain, called “DNA-

binding motifs”, into the major α Helix groove. Turn α Helix 3.4 nm

Several common DNA-binding 90° motifs are shared by many different regulatory proteins. One example is the helix-turn-helix motif: Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 15 16

Objective 3 Once the DNA-binding motif is inserted into the major groove, it examines the chemical groups that project into the groove. Since each of the 4 possible base pairs has a different set of chemical groups that extend into the groove, the regulatory protein can determine the base pair sequence by examining these groups: 17 18

3 Objective # 4 Objective 4 Gene regulation relies on both negative Distinguish between negative and positive control systems: control systems and positive ¾ In negative control systems, the regulatory protein is a repressor which control systems. binds to DNA and blocks transcription. ¾ In positive control systems, the regulatory protein is an activator which binds to DNA and promotes transcription. 19 20

Objective 4 Objective 4 We will look at 3 regulatory systems. 2) In regulation by induction: 1) In regulation by repression: ¾ The repressor alone can bind to ¾ The repressor alone cannot bind to DNA and block transcription. DNA and block transcription. ¾ When an inducer binds to the ¾ However, when a co-repressor binds repressor, the repressor is to the repressor, the repressor is inactivated and can no longer bind activated. Once activated, the repressor can bind to DNA and to DNA and block transcription. block transcription. 21 22

Objective 4 3) In regulation by activation: ¾ The activator alone cannot bind to DNA and stimulate transcription. ¾ When an inducer joins with the activator, the activator can bind to DNA and stimulate transcription.

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4 Objective # 5 Objective 5 Prokaryotes can quickly turn production Describe the process of gene of specific proteins on or off in regulation in prokaryotic cells, response to environmental conditions. and provide examples of both This rapid response is facilitated by 3 negative and positive important mechanisms: transcriptional control in a ¾ simultaneous transcription and translation prokaryotic cell such as E. coli. ¾ short-lived m-RNAs ¾ 25 operons 26

Objective 5 Objective 5

Operons: ¾ A single promoter serves all 5 genes. ¾ In prokaryotes, functionally related Recall that the promoter is the region genes are often located next to each where RNA polymerase binds to other and are transcribed as a unit. DNA and begins transcription ¾ For example, in E. coli 5 different ¾ The genes are transcribed as a unit, enzymes are needed to synthesize the producing one long mRNA molecule amino acid tryptophan. The genes (polycistronic mRNA) which contains that code for these enzymes are the code to make all 5 enzymes.

located together. 27 28

Objective 5 Objective 5

¾ There is also a single regulatory switch, called the operator. Transcription of the 5 coding genes in the tryptophan operon is blocked ¾ The operator is positioned within the when a transcriptional repressor binds promoter, or between the promoter and to the operator. the protein coding genes. It controls access of RNA polymerase to the genes. The repressor binds to the operator only when it is activated by the ¾ All together, the operator, promoter, presence of tryptophan: and the genes they control is called an

operon. 29 30

5 Tryptophan repressor The tryptophan Objective 5 repressor is activated when it joins to tryptophan Because transcription of the coding genes in the tryptophan operon can be Tryptophan blocked by a repressor, it is an example of negative transcriptional DNA control:

Objective 5

Other regulatory proteins act as transcriptional activators by enhancing the ability of RNA polymerase to join with the promoter. For example, CAP (catabolite activator protein) stimulates transcription of genes that allow E. coli to use other food sources when glucose is not present: 33 34

Objective 5 cAMP binds to CAP, altering its shape so ¾ Low levels of glucose lead to high it can bind to DNA:

levels of cAMP. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ¾ The cAMP binds to CAP, altering its DNA shape so it can bind to DNA near any of several promoters. This bends the DNA, making it easier for RNA polymerase to join with the promoter and initiate transcription: CAP cAMP 35 36

6 Objective 5 lac Operon of E. coli The lactose operon is present in Gene for repressor protein Gene for E. coli. It controls the production of permease CAP-binding Operator Promoter site 3 enzymes needed to digest lactose for I gene Promoter for Gene for Gene for (a disaccharide made of glucose and lac operon β-galactosidase transacetylase P I CAP Plac O I Z galactose). Y A The lac operon consists of a coding Regulatory region Coding region region and a regulatory region: lac control system

Objective 5 Objective 5

Since glucose is the preferred food for E. coli, it makes sense to produce the 3 How lactose affects the lac operon: enzymes needed to digest lactose only ¾ When lactose is absent, the lac when lactose is present AND glucose repressor binds to the operator and is absent. blocks transcription of the genes Therefore, the lac operon is affected needed to digest lactose: by the levels of both lactose and glucose in the cell.

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When lactose is absent, the lac repressor Objective 5 binds to the operator and blocks transcription: ¾ When lactose is present, a metabolite lac repressor polypeptide mRNA lac Repressor of lactose called allolactose binds to (No lactose present) CAP–binding site the repressor and inactivates it. lac repressor cAMP Therefore, the repressor can no longer lac repressorCAP bind to the operator and block gene Promoter for transcription: lac operon DNA ZYA Operator RNA polymerase is blocked by the lac repressor No transcription Enzymes to degrade lactose not produced 41 42 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

7 When lactose is present, a metabolite of lactose called allolactose binds to the repressor and inactivates it:

β-Galactosidase Allolactose (inducer) (lactose present) mRNA Transacetylase Translation Permease

lac Repressor Enzymes to degrade lactose produced cannot bind to DNA mRNA

Objective 5 When glucose is low, CAP binds to DNA How glucose affects the lac operon: and stimulates transcription: ¾ When the glucose level is low, cAMP DNA CAP- binding is high. The cAMP binds to CAP, site Allolactose altering its shape so it can bind to Repressor will mRNA not bind to DNA DNA near any of several promoters. cAMP–CAP This bends the DNA, making it easier binds to DNA CAP cAMP Glucose level is A for RNA polymerase to join with the cAMP Y low cAMP is high Z cAMP activates promoter and initiate transcription CAP by causing a RNA polymerase is not blocked conformation change and transcription can occur

Objective 5 When glucose is high, CAP does not bind ¾ When the glucose level is high, cAMP is to DNA but the lac repressor does:

low. Therefore, CAP is not activated Glucose is available and transcription is not stimulated. cAMP level is low ¾ In addition, high glucose inhibits Repressor binds to DNA A transport of lactose into the cell. Low CAP does not bind lactose allows the lac repressor to bind Y to the operator and block transcription. Z

This mechanism, called inducer Effector site is empty, and there is no RNA polymerase is exclusion, is the primary way glucose conformation change blocked by the lac repressor

8 Objective # 6

Describe the process of gene regulation in eukaryotic cells and compare it with gene regulation in prokaryotic cells.

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Objective 6 Objective 6

In eukaryotes, gene regulation is more complex and more likely to involve In prokaryotes, functionally related posttranscriptional controls. genes are grouped together to form However, like in prokaryotes, operons. transcriptional controls are still the primary method of gene regulation in Eukaryotes lack operons. eukaryotes, and they will be the main focus of our discussion.

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Objective 6 lac Operon of E. coli Gene for repressor protein Gene for In addition, in prokaryotes regulatory permease CAP-binding Operator molecules control transcription by Promoter site for I gene Promoter for Gene for Gene for binding to DNA sequences within or lac operon β-galactosidase transacetylase

P I CAP Plac O very close to the promoter. I Z Y A This limits the number of regulatory Regulatory region Coding region molecules that can affect transcription: lac control system

9 Objective 6 Objective 6

In eukaryotes, on the other hand, a The regulatory molecules involved in much larger number of regulatory binding RNA polymerase to the molecules are required in order for promoter and initiating transcription in RNA polymerase to bind to the eukaryotes are called transcription promoter and initiate transcription. factors. Because many more regulatory Transcription factors can be either molecules are involved, more complex general or specific. regulation is possible.

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Objective 6 Objective 6 General transcription factors are also Specific transcription factors include called basal factors. They bind to the activators, coactivators, and repressors. promoter to form an initiation complex. Activators bind to regulatory sequences The initiation complex captures and on the DNA called enhancers that may stabilizes RNA polymerase on the be located far away from the promoter. promoter. This initiates transcription at Because enhancers can be scattered the basal level. The basal level can be anywhere in the genome, many different activators can affect the transcription of increased or decreased through the a single gene. action of specific transcription factors. 57 58

General Objective 6 Transcription RNA polymerase Activator factor Transcribed region

Activators act to increase the rate of Promoter transcription by interacting with the Enhancer TATA box general transcription factors (also called basal factors) either directly or with the help of coactivators. mRNA synthesis Two regulatory proteins (an activator and a general transcription factor) are needed to bind RNA polymerase to the promoter 59 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

10 Objective 6 Objective 6

In more complex systems, several A third group of specific transcription activators may interact with many factors are called repressors. These different coactivators and basal factors proteins bind to regulatory sequences in order to bind RNA polymerase to called silencers located adjacent to or overlapping enhancers. the promoter and initiate transcription. Binding of a repressor to a silencer Anything that affects the availability of blocks binding of the activator to the a particular activator, coactivator or enhancer and therefore inhibits basal factor will affect the rate of transcription: transcription. 61 62

Objective 6

Alterations to chromatin structure may also help regulate gene transcription in eukaryotes. For example, addition of acetyl groups (called acetylation) to histone proteins can alter chromatin structure, making genes accessible for transcription:

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Nucleosomes block the binding of RNA polymerase II to the promoter Amino acid histone tail

Condensed solenoid

N-terminus

Addition of acetyl groups to histone tails remodel the solenoid so that DNA is accessible for transcription

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Although less common than transcriptional control of gene expression, various types of posttranscriptional control may also occur in eukaryotes:

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