Chapter 16

list, describe, and give examples of the different mechanisms of gene regulation

1. *alteration of DNA or chromatin structure* - determines which sequences are available for transcription or the rate of transcription - eg. DNA methylation 2. regulation at the *transcriptional level* - most gene regulation occurs here*** 3. *mRNA processing* - eg. 5' cap, poly (A) tail, and intron removal - these modifications determine mRNA stability, movement of mRNA into the cytoplasm, whether mRNA can be translated, the rate of translation, and the AA sequence of the protein produced 4. regulation of *mRNA stability* - the amount of protein produced depends on the amount of mRNA translated, which depends on the rate at which the mRNA is degraded while waiting to be transcribed, which depends on the stability of the mRNA. 5. regulation at the *translational level* - requires enzymes, protein factors, RNA molecules, and the availability of AA's. All of these factors determine the rate at which proteins are produced. (and certain proteins are responsible for regulating gene expression, eg. DNA binding proteins) - translation can be affected by sequences in mRNA such as those in the 5' and 3' untranslated regions 6. *post-translational modification* of proteins - these modifications affect whether the proteins become active.

In the process of attenuation in trp operons, when tryptophan levels are *high,* the ribosome covers region ____ while region 3 is being transcribed and the ____ hairpin forms. When tryptophan levels are *low,* the ribosome covers region ____ while region 3 is being transcribed and the ____ hairpin forms.

2; 3+4 (attenuator) 1; 2+3

What is attenuation?

Attenuation is an additional level of control used by some operons that affects the continuation of transcription rather than initiation. In attenuation, transcription begins at the transcription start site, but is terminated before the RNA polymerase even reaches the first structural gene. It takes place in a number of operons that encode enzymes participating in the biosynthesis of amino acids (eg. tryptophan synthesis).

describe the structure and function of DNA binding proteins

DNA binding proteins bind to certain regulatory sites on DNA to affect the expression of a gene,. These regulatory proteins have discrete functional parts called *domains* containing 60-90 AA that are responsible for binding to DNA by forming *H bonds* with the bases or *interacting* with the sugar or phosphate backbone (no covalent bonds are formed). They can also bind to other regulatory proteins. Only a few of the AA's actually make contact with the DNA. Most of these protein bind dynamically, meaning they transiently (for a short time) bind and unbind DNA and other regulatory proteins, thus, other molecules can compete with them for regulatory sites on the DNA. DNA binding proteins can be grouped according to characteristic structures within the binding domain called *motifs* which are simple structures that can fit into the major groove of the DNA double helix (eg. the helix-turn-helix motif, zinc finger motif, and the leucine zipper motif)

______ first described the "operon model" for the genetic control of lactose metabilism in E coli in 1961. Their work, and lead to the establisment of the operon as the basic unit of transcriptional control in bacteria. They analyzed the interactions of mutations that interfered with the normal regulation of lactose metabolism

Francois Jacob and Jaques Monod

Describe the basic *transcriptional* control mechanisms of operons

In negative control, a regulatory protein is a repressor, which binds to DNA and inhibits transcription. In positive control, a regulatory protein is an activator, stimulating transcription. Operons can either be inducible or repressible. *Inducible operons* are those in which transcription is normally off (not taking place) - something must happen to induce transcription. *Repressible operons* are those in which transcription is normally on - something must happen to repress transcription.

explain lac operator (lacO) gene mutations

J&M mapped a class of constitutive mutations called *lacOᶜ* (superscript c stands for constitutive). The lacOᶜ mutations altered the sequence of DNA at the operator so that the repressor protein was no longer able to bind it. lacOᶜ is *dominant* over lacO⁺... A: [lacI⁺ lacOᶜ lacZ⁺ / lacI⁺ lacO⁺ lacZ⁺] → constitutive synthesis of β-gal Another analysis of a partial diploid revealed that the lacO gene is *cis-acting,* affecting only genes on the same DNA molecule... B: [lacI⁺ lacO⁺ lacZ⁻ / lacI⁺ lacOᶜ lacZ⁺] → constitutive β-gal synthesis regardless in presence OR absence of lactose. C: [lacI⁺ lacO⁺ lacZ⁺ / lacI⁺ lacOᶜ lacZ⁻] → β-gal produced only in the presence of lactose. in genotype B, the lacOᶜ mutation and the functional lacZ⁺ were on the same DNA molecule, but in genotype C, the lacOᶜ mutation and the lacZ⁺ gene were on different DNA molecules. ALL lacO mutations are cis-acting and only affect genes to which they are physically connected. (think about it - how would an operator on one chromosome affect the transcription of lac genes on another? lacO doesn't even encode a protein its just a DNA binding site)

explain lac operon *structural* gene mutations

Jacob and Monod mapped out mutations of *lacZ* and *lacY* structural genes that altered the AA sequences of the proteins encoded by them (β-gal and permease). These mutation clearly affected the *structure* of the proteins, but NOT the regulation of their synthesis. They established that mutations of the lacZ and lacY genes were independent and usually affected only the product of the gene in which the mutation occurred. The following genotype is of a cell with lacZ⁺ lacY⁻ on the bacterial chromosome and lacZ⁻ lacY⁺ on the plasmid - this is how you write a genotype of a partial diploid: [lacZ⁺ lacY⁻/ lacZ⁻ lacY⁺] → produced β-gal and permease in the presents of lactose. A single functional β-gal gene (lacZ⁺) was sufficient to produce β-gal; whether the functional β-gal gene was coupled to a functional (lacY⁺) or a defective (lacY⁻) permease gene make no difference. The same was true for that of the lacY⁺ gene.

explain what an operon is

Many bacterial genes that have *related functions* are clustered together and under the same promotor, and are often transcribed together in a single mRNA molecule. A group of *bacterial* structural genes that are transcribed together, along with their promotor and additional sequences that control their transcription, is called an *operon.* The operon regulates expression of the structural genes by controlling transcription. Fig. 16.3 - RNA polymerase binds to the promotor and transcribes gene a, gene b, and gene c into an mRNA molecule that translates into enzymes A, B, and C. These enzymes, in this example, carry out a series of reactions in a biochemical pathway (eg glycolysis). A *regulator gene* helps control the expression of the structural genes of the operon by increasing or decreasing transcription. the regulator gene is not considered part of the operon. It has its own promotor and encodes a small protein called a *regulator protein.* This regulator protein binds to a region of the operon called the *operator* and affects whether transcription can take place. The operator usually overlaps the 3' end of the promotor and sometimes the 5' end of the first structural gene.

explain lac operon *regulator* gene mutations

The lacI gene codes for the repressor protein (recall that repressors are regulator proteins). Mutations in the lacI gene (repressor gene) affect the production of both β-gal and permease. Some of the mutations of lacI seen were constitutive, causing the lac proteins (β-gal and permease) to be produced all the time whether lactose was present or not. Such mutations in the regulator genes were designated lacI⁻. Construction of partial diploids demonstrated that.. - a lacI⁺ gene is *dominant* over a lacI⁻ gene (a single copy of lacI⁺ is sufficient to bring about normal regulation of protein production) - lacI⁺ can be *trans acting.* (a single copy of lacI⁺ is able restore normal control to an operon with a defective lacI⁻ gene even if the operon is located on a different DNA molecule.) [ lacI⁺ lacZ⁻ / lacI⁻ lacZ⁺ ] → functions normally, synthesizing β-gal only when lactose is present. *superrepressors (lacIˢ)* are mutations in the lacI gene that produces defective repressor proteins with altered inducer-binding sites which means inducers are unable to inactivate the repressor, and the repressor remains attached to the operator preventing transcription of the lac genes. lacIˢ mutations are *dominant* over lacI⁺ [ lacIˢ lacZ⁺ lacY⁺ / lacI⁺ lacZ⁺ lacY⁺ ] → unable to synthesize either β-gal or permease, whether or not lactose was present.

explain negative control of repressible operons

The regulator protein that acts on a negative repressible operon is [like in negative inducible operons] also a repressor, except it is synthesized in an *inactive* form (it cannot by itself bind to the operator.) Thus, there is an open spot on the promotor for RNA polymerase to bind to, and transcription of the operon's structural genes takes place. To turn transcription off, something must happen to make the repressor active. When a small molecule called a *corepressor* is present, it binds to the repressor and makes it capable of binding to the operator. in the example in the book, the product (U) of the metabolic pathway controlled by the negative repressible operon is the corepressor. As long as the level of product U is high, it is available to hind to the repressor and activate it, preventing transcription of the enzymes that synthesize product U, and no more product U is made (negative feedback!). When all of product U is used up, the repressor is no longer activated by product U (the corepressor) and and cannot bind to the operator.

explain the trp operon in E. coli

The trp operon is unusual in that it is regulated both by repression and attenuation (whereas most operons are regulated by one of these mechanisms and not both). REPRESSION MECH: The tryptophan (trp) operon in E. coli controls the biosynthesis of tryptophan and is an example of a *negative repressible operon.* Whereas the lac operon contains 3 structural genes, the trp operon contains 5 (trpE, trpD, trpC, trpB, trpA.. in that order) that produce the components of three enzymes (two of the enzymes contain two polypeptide chains). These enzymes convert a precursor called chorismate into tryptophan. When tryptophan *(the corepressor)* levels are low (i.e. more is needed), RNA polymerase binds to the promotor and transcription occurs, thus tryptophan is produced. Some distance from the trp operon is a regulator gene, *trpR,* which encodes a *repressor* protein that is normally *inactive.* Like the lac operon repressor, trpR has two binding sites: one that binds to the operator and another that binds to tryptophan. When tryptophan levels are high, the repressor binds with tryptophan which causes a conformational change in the repressor that activates it (makes it able to bind to the operator). When the operator is occupied by the repressor, RNA polymerase cannot bind to the promotor. ATTENUATION MECH: The first structural gene of trp operon, trpE, has a long 5' untranslated region that is transcribed but does not encode proteins. Once transcribed, its mRNA strand contains 4 regions with the following complementaries: region 1 complements region 2, region 2 complements region 3, and region 3 complements region 4. As a result, one hairpin is formed by the base pairing between regions 1 and 2, and another is formed by the base pairing of regions 3 and 4, and the 3+4 hairpin is followed by a string of uracils. Its no coincidence that the structure of a bacterial terminator includes a hairpin followed by a string of uracils. This secondary structure is indeed a terminator of transcription and is called an *attenuator.* The attenuator forms when tryptophan levels are *high,* causing transcription to be terminated before the trp structural genes can be transcribed. However, when tryptophan levels are *low,* a hairpin is created by the base pairing of regions 2 and 3. This hairpin is not followed by a string of uracils, therefore it does not terminate transcription, and RNA polymerase continues past it and transcribes the structural genes. Because it prevents termination, the 2+3 structure is called an *antiterminator.*

explain lac promotor mutations

These mutations, designated *lacP⁻,* interfere with the binding of RNA polymerase to the promotor, inhibiting transcription of lac proteins whether or not lactose is present. Like lacO genes, lacP genes do not encode proteins, so they are cis-acting and only affect transcription of lac proteins on the same DNA molecule. [ lacI⁺ lacP⁺ lacZ⁺ / lacI⁺ lacP⁻ lacZ⁺ ] → exhibits normal synthesis of β-gal [ lacI⁺ lacP⁻ lacZ⁺ / lacI⁺ lacP⁺ lacZ⁻ ] → fails to produce β-gal whether or not lactose is present

explain negative control of inducible operons

These types of operons are said to be "inducible." The regulator gene for a negative inducible operon encodes a *repressor* protein synthesized in an *active* form that readily binds to the operator. Because the operon's operator site overlaps its promotor, it physically blocks RNA polymerase from binding to the promotor thus preventing transcription. For transcription to be induced, a small molecule called an *inducer* must be present and bind to the repressor so that the repressor cannot bind to the operator. Regulatory proteins frequently have two binding sites: one to bind DNA and another to bind molecules such as inducers. When a repressor binds an inducer, the repressor undergoes a change in shape, rendering it inactive and unable to bind to DNA. If the inducer is absent, the repressor binds to the operator and the structural genes are not transcribed. MY example: glycolysis (glucose is the inducer, and when present, transcription of hexokinase, PFK, etc are transcribed to act on the glucose)

proteins which change shape upon binding to another molecule are called ____

allosteric proteins eg. repressor proteins

Which mechanisms of gene regulation are used in eukaryotes but are largely absent in prokaryotes?

alteration of DNA/chromatin structure and mRNA processing

why is transcription a particularly important level of gene regulation in both bacteria and eukaryotes?

because transcription is the first step in the process of information transfer from DNA to protein. It is more efficient to regulate early on to save resources

some structural genes, particularly those that encode essential cellular functions (often called housekeeping genes) are expressed continuously and are therefore said to be _____. These genes are *not regulated.*

constitutive

the regulator protein that act on a negative repressible operon is synthesized as a. an active activator b. an inactive activator c. an active repressor d. an inactive repressor

d. an inactive repressor

describe positive control in E. coli lac operons

glucose is metabolized by bacteria preferentially over other sugars because it is more energy-efficient, so when glucose is available, genes that participate in the metabolism of other sugars are turned off through a process called *catabolite repression.* Efficient transcription of the lac operon takes place only if lactose is present and glucose is absent. When glucose is present, catabolite repression occurs by a *positive control* mechanism where an activator protein (the opposite of a repressor protein) called *catabolite activator protein* (CAP) binds to a sequence (we'll call it the CAP site) within or slightly upstream of the lacP. This increases the efficiency of the binding of RNA polymerase to the promotor. However, before CAP can bind to the DNA it must form a complex with cAMP. The availability of cAMP depends on glucose levels. When glucose levels are high, cAMP levels are lowered, and vice versa. high glucose levels → low cAMP levels → no CAP-cAMP complex is formed → CAP cannot bind to the CAP site → RNA polymerase cannot efficiently bind to the DNA → little transcription of lac genes occur → lactose metabolism is repressed.

describe positive control of operons

in positive control, the regulatory protein is an *activator protein* (as opposed to a repressor protein). It binds to DNA (usually as a site other than the operator) and stimulates transcription. For example, in the case of the lac operon, the catabolite activator protein (CAP) binds to the promotor and increases the efficiency with which RNA polymerase can bind to the promotor and transcribe the structural genes.

____ operons usually control the proteins that carry out catabolic processes, while ______ operons usually control proteins that carry out anabolic processes

inducible - this makes sense because the proteins are not needed unless the substrate (which is broken down by the proteins) is present. repressible - this makes sense because the product produced by the proteins is always needed by the cell, thus, their transcription is usually on and only turned off when there are adequate amounts of the product

in the trp operon, what happens to the trp repressor in the absence of tryptophan?

it cannot bind to the operator, and transcription takes place

describe lactose metabolism in E. coli

lactose is metabolized by E. coli in the mammalian gut, but it must be actively transported across the E. coli cell membrane by the protein *lactose permease.* To be used as energy, the lactose must first be broken down into glucose and galactose via the enzyme β-galactosidase. β-gal can also convert lactose into allolactose. A third enzyme, thiogalactoside transacetylase, is also produced by the lac operon but its function in lactose metabolism is unclear.

indicate with a (+) or (-) the synthesis of β-gal and permease in the presence, and in the absence of lactose in an E. coli strain with the following genotype: lacI⁺ lacP⁺ lacO⁺ lacZ⁻ lacY⁻ / lacI⁻ lacP⁺ lacO⁺ lacZ⁺ lacY⁺

lactose present: β-gal + permease + lactose absent: β-gal - permease -

indicate with a (+) or (-) the synthesis of β-gal and permease in the presence, and in the absence of lactose in an E. coli strain with the following genotype: lacI⁺ lacP⁺ lacO⁺ lacZ⁺ lacY⁺

lactose present: β-gal + permease + lactose absent: β-gal - permease - the lac operon of E.coli is an example of a negative inducible operon, so in order for transcription to occur there must be an inducer present to bind to the repressor so that it releases from the operator.

indicate with a (+) or (-) the synthesis of β-gal and permease in the presence, and in the absence of lactose in an E. coli strain with the following genotype: lacI⁺ lacP⁺ lacOᶜ lacZ⁻ lacY⁺

lactose present: β-gal - permease + lactose absent: β-gal - permease + This is not a partial diploid, therefore lacZ⁻ is the only form of the lacZ gene in the entire organism, so β-gal will not be produced under any conditions. lacOᶜ means the operator cannot bind to a repressor at all, so the promotor is always available for RNA polymerase, so permease will be constitutively produced whether or not lactose is present

indicate with a (+) or (-) the synthesis of β-gal and permease in the presence, and in the absence of lactose in an E. coli strain with the following genotype: lacI⁺ lacP⁻ lacO⁺ lacZ⁺ lacY⁻

lactose present: β-gal - permease - lactose absent: β-gal - permease - a mutation in the promotor sequence means no transcription, period.

in the trp operon, why does transcription terminate prematurely when tryptophan levels are high, but continue when tryptophan levels are low?

recall that in prokaryotes, transcription and translation are coupled, so while transcription is taking place at the 3' end of the mRNA, translation is initiated at the 5' end of the mRNA. RNA polymerase begins transcribing the DNA, producing region 1 of the 5' UTR of trpE (the first structural gene). Closely following RNA polymerase, a ribosome binds to a ribosome binding site directly upstream from a start codon in the 5' UTR of the newly transcribed trpE mRNA and begins to translate it. Meanwhile, RNA polymerase is transcribing region 2. Although regions 1 and 2 are complementary, they cannot form a hairpin at this point because the ribosome is still translating region 1. RNA polymerase begins to transcribe region 3. Meanwhile, the ribosome reaches the two trp codons (UGGUGG) that are characteristic of region 1. Here, translation is either continued or stalled depending on availability of tryptophan in the cell, because a tRNA charged with tryptophan is required for the translation of the UGG codons. So when levels of tryptophan are high, there will be tRNAs with tryptophan and translation will continue, and when tryptophan levels are low, tRNAs with tryptophan will be scarce or unavailable and translation will be stalled at this point. Whether translation is stalled or not, transcription continues on to region 4. If translation is stalled in region 1 due to *low* tryptophan levels, *transcription gets ahead of translation,* and regions 2 and 3 are free to form a hairpin, making it impossible for the 3+4 hairpin (the attenuator) to form. This 2+3 hairpin does not terminate transcription, so transcription of the structural genes continues (there is another start codon between region 4 and the protein-encoding region of the trp gene, so another ribosome can begin translation here). If tryptophan levels are *high,* the ribosome does not stall at the two trp codons because tryptophan is abundant and tRNAs charged with tryptophan are available. Because translation does not stall, *translation can keep up with transcription.* As translation moves past region 1, the ribosome partly covers region 2 while RNA polymerase completes transcription of region 3. The ribosome on region 2 prevents the 2+3 hairpin from forming. Although translation continues, it is still slower than transcription, so once transcription moves along region 4, the ribosome is still on region 2, so regions 3 and 4 are able to form a hairpin as region 4 is translated. This forms the attenuator, and transcription terminates just beyond region 4. The structural genes are not transcribed, so no tryptophan-synthesizeing enzymes are produced.

DNA binding proteins are encoded by ____ gene sequences

regulatory

DNA sequences that are not transcribed but still play a role in regulating genes and other DNA sequences are called ____ and affect the expression of DNA sequences to which they are physically linked.

regulatory elements

In all organisms, there are 2 major types of DNA sequences that undergo transcription. ____ genes encode proteins that are used in metabolism or biosynthesis or that play a structural role in the cell. The products of ____ genes - either RNA or proteins - interact with other DNA sequences and affect the transcription or translation of those sequences.

structural; regulatory

describe the regulation of the lac operon in E. coli

the lac operon in E. coli is an example of a negative inducible operon. The enzymes β-gal, permease, and transacetylase are encoded by adjacent structural genes in the lac operon and have a common promotor (lacP in fig 16.7a). β-gal is encoded by the lacZ gene, permease by the lacY gene, and transacetylace by the lacA gene. When lactose is absent from the medium in which E. coli grows, few molecules of each protein are made. If lactose is added AND glucose is absent, the rate of synthesis of all 3 proteins skyrockets within 2-3 mins. This simultaneous transcription of lacZ, lacY, and lacA exemplifies *coordinate induction*, the simultaneous synthesis of several proteins stimulated by a specific molecule, the inducer. Although lactose may appear as the inducer here, the inducer is actually allolactose. Upstream of lacP is a regulator gene called lacI (thats a capital i) which has its own promotor (PI). The lacI gene encodes a repressor with 2 binding sites: one that binds to DNA and one that binds to allolactose. In the absence of lactose (and therefore allolactose), the repressor binds to the lac operator, lacO. lacO overlaps the 3' end of the lacP and the 5' end of lacZ. When the repressor of bound to lacO, the binding of RNA polymerase is blocked. When lactose is present, some of it is converted to allolactose which binds to the repressor rendering the repressor inactive (unable to bind to operator) and it detaches from the operator. RNA polymerase then binds to the promotor and transcription of lacZ, lacY, and lacA takes place, and the 3 corresponding lac proteins are produced. repression never completely shuts down transcription. Even when the active repressor is bound to the operator, there is a low level of transcription, so there are always a few molecules of permease (required to transport lactose into the cell), β-gal (required to convert lactose into allolactose) and transacetylase.

describe positive and negative control

the regulation of gene expression by STIMULATING gene expression is called positive control, whereas the regulation by gene INHIBITION is called negative control

What is coordinate induction?

the simultaneous synthesis of several proteins stimulated by a specific molecule.

describe basis of Jacob and Monod's experiment with lac mutations

they used partial diploid strains of E. coli which possessed the full bacterial chromosome plus an extra piece of plasmid DNA added via conjugation. The recipient cell contained the lac operon on both the plasmid DNA and the bacterial chromosome. They tested different combinations of lac operon mutations and found that some pars of the lac operon are cis acting (able to control the expression of genes only on the same piece of DNA), whereas other parts are trans acting (able to control the expression of genes on other DNA molecules.)