Genetics 15.1-16.3

complementation and mapping analyses of Lac- mutants

-Lac- mutants are bacterial cells unable to utilize lactose. -using complementation analysis, researchers showed that the inability to break down lactose resulted from mutations in two genes: lacZ, which encodes B-galactosidase, and lacY, which encodes Lac permease -they also discovered lacA, which encodes a transacetylase enzyme that adds an acetyl (CH3CO) group to lactose and other sugars. -genetic mapping shows the three genes in a tightly linked cluster, in the order lacZ-lacY-lacA -bc lacA gene product is not required for the breakdown of lactose, most studies of lactose utilization have focused only on lacZ and lacY

positive regulation of the lac operon

-high level expression of the lac operon requires that a positive regulator, the CRP-cAMP complex, be bound to a site near the promoter. the complex is a dimer, and the site to which it is bound contains rotationally symmetrical DNA sequences. the CRP-cAMP complex contacts RNA polymerase directly to help in transcription initiation -CRP-binding sites consist of two recognition sequences pointing in opposite directions, each able to bind one subunit of the CRP dimer. -this ex again stresses the importance both of the multimerization of DNA-binding protein subunits and of the clustering of their corresponding binding sites in the vicinity of promoters

two types of termination signals found in prokaryotes

-in Rho-dependent termination, a protein factor called Rho recognizes a sequence in the newly transcribed mRNA and terminates transcription by binding to the RNA and pulling it away from the RNA polymerase enzyme. -in Rho-independent termination, a sequence of bases in the RNA forms a secondary structure, known as a hairpin loop, that serves as a signal for the release of RNA polymerase from the completed RNA.

chromatin immunoprecipitation-sequenceing (ChIP-Seq)

a powerful new technology for finding all the target genes of a particular transcription factor within the entire genome of a particular cell

allosteric protein

a protein that undergoes a reversible change in conformation when bound to another molecule (in this case, allolactose)

eukaryotes: enhancer

a regulatory site that can be quite distant-- up to tens of thousands of nucleotides away-- from the promoter. important for transcription -binding of proteins to enhancers can augment or repress basal levels of transcription

riboswitches

allosteric RNA leaders that bind small molecule effectors to control gene expression -leaders that act as riboswitches have a region called the aptamer that binds a particular effector directly. -riboswitches also have a second region, called the expression platform, which controls gene expression by altering its stem-loop structures in response to the aptamer configuration -in some riboswitches, the expression platform controls the termination of transcription -in others, the expression platform regulates translation by blocking or unblocking the ribosome binding site

positive and negative regulators

although pos and neg regulators obviously have opposite effects on transcription, you should keep in mind that most regulators of both types work through their effects on RNA polymerase. many neg regulators, such as the Lac repressor, prevent initiation by blocking the functional binding of RNA polymerase to the promoter. pos regulators, by contrast, usually act by establishing a physical contact with RNA polymerase that attracts RNA polymerase to the promoter or that keeps RNA polymerase bound to DNA longer so that it initiates transcription more frequently

repressible regulation

anabolic pathways require repressible regulation: this means the pathway should be turned on only when the cell does not have enough of the needed end product. if the end product is present in sufficient quantities, the pathway should be turned off- repressed- so the cell does not waste resources trying to make molecules it already has.

indirect repressors

many regulatory proteins prevent transcription initiation indirectly, not by recruiting corepressors, but instead by interfering with the function of activators -some repressors can compete with activators for access to an enhancer bc the binding sites of both overlap -in quenching, a protein can bind the activation domain of an activator bound to an enhancer and thereby prevent the activator from functioning -an activator can be sequestered in the cytoplasm when bound to an indirect repressor -formation of a nonfunctional indirect repressor/ activator heterodimers can prevent the formation of functional activator homodimers

catabolic pathways

metabolic pathways in which complicated molecules are broken down for the use of the cell; ex of catobolic pathways are those that break down sugars to provide cells with energy and carbon atoms.

2 mutations that allow expression of the lac operon even when the inducer is not present

-constitutive operator (o^c) mutants (repressor cannot bind to a changed sequence in the operator) -constitutive lacI- mutants (encode mutant repressor that cannot bind to operator) -both mutations prevent repression

helix-turn-helix motif

-Xray crystallographic studies revealed that the DNA-binding domain of Lac repressor subunits has a characteristic 3D structure: two alpha-helical regions are separated by a turn. this helix-turn-helix motif fits well into the major groove of the DNA -the HTH motif is found in hundreds of DNA-binding proteins, not only in bacteria but also in eukaryotic cells. -one of the alpha helixes in each HTH-containing transcription factor carries unique amino acids that recognize a specific DNA sequence of nucleotides. as a result, various HTH-containing proteins can bind to unique DNA sequences. in bacteria, this means that different HTH-containing transcription factors interact with different operators to regulate different genes and operons.

RNA leader sequence

-all bacterial mRNAs begin with an untranslated region called the 5' UTR, or RNA leader sequence. -through complementary base pairing, many RNA leaders form secondary structures called stem loops. -the stem loops can terminate transcription of the rest of the mRNA prematurely, or they can prevent translation by blocking access of the mRNA to the ribosome binding site -these RNA leaders are allosteric in that they can alter their stem-loop structures and thus their function in response to a wide variety of environmental cues.

tryptophan as a corepressor

-anabolic pathways require repressible regulation -E. coli tryptophan (trp) operon is a group of five genes-- trpE, trpD, trpC, trpB, and trpA-- required for biosynthesis of the amino acid tryptophan. -max expression of the trp genes occurs when trp is absent from the growth medium. -the trp operon is controlled in part at the level of transcription initiation through the action of a repressor protein that is the product of the trpR gene. -tryptophan functions as a corepressor for the TrpR repressor protein -tryptophan is .a small molecule effector whose binding to the TrpR protein allows this negative regulator to bind to DNA and inhibit transcription of the genes in the trp operon. -the binding of tryptophan to the TrpR repressor causes an allosteric alteration in the repressor's shape, and only with this alteration can the TrpR protein bind to the operator site. -mutations in the trpR gene that change either the protein's tryptophan-binding domain or its DNA-binding domain destroy the TrpR repressor's ability to associate with DNA, and both types of mutations therefore result in the constitutive expression of the trp genes even when tryptophan is present in the growth medium.

translation in prokaryotes begins before transcription ends

-bc no membrane encloses the bacterial chromosome, translation of the RNA message into a polypeptide can begin while mRNA is still being transcribed. ribosomes bind to special initiation sites at the 5' end of the reading frame (the ribosome binding site) while transcription of the downstream regions of the RNA is still in progress. signals for the initiation and termination of translation are distinct from signals for the initiation and termination of transcription. -bc prokaryotic mRNAs are often polycistronic (they contain open reading frames for several different proteins), ribosomes can initiate translation at several positions along a single mRNA molecule.

attenuation

-in a series of experiments, Charles Yanofsky found that this repressor-independent change in trp operon expression involves the production of alternative trp operon transcripts -sometimes initiation at the promoter leads to transcription of a truncated mRNA about 140 bases long containing only the RNA leader but none of the structural genes. -at other times, transcription continues beyond the end of the leader sequence to produce a full operon-length transcript -in analyzing why some mRNAs terminate before they can transcribe the structural trp genes, while others do not, the researchers discovered attenuation: control of gene expression by RNA leader-mediated premature termination of transcription -whether or not transcription terminates depends on how the translation machinery reads the secondary structure of the RNA leader

CRP protein and the response to glucose

-inside bacterial cells, the small nucleotide known as cAMP binds to a protein called cAMP receptor protein, or CRP -the binding of cAMP to CRP enables CRP to bind to DNA in the regulatory region of the lac operon near the promoter -when bound to DNA, CRP helps recruit RNA polymerase to the promoter by making physical contacts with the polymerase enzyme; in essence, the DNA binding of CRP increases the ability of RNA polymerase to transcribe the lac genes -CRP thus functions as a positive regulator that enhances the transcriptional activity of RNA polymerase at the lac promoter, while cAMP is an effector whose binding to CRP enables CRP to bind to DNA neat the promoter and carry out its regulatory function. -an effector is a small molecule that binds to an allosteric protein or an RNA molecule and causes a conformational change (an inducer such as allolactose is, like cAMP, an effector that activates gene expression) -glucose controls the amount of cAMP in the cell indirectly by decreasing the activity of adenyl cyclase, the enzyme that converts ATP into cAMP. thus, when glucose is present, the level of cAMP remains low; when glucose is absent, cAMP synthesis increases. as a result, when glucose is present in the culture medium, there is little cAMP available to bind to CRP and therefore little induction of the lac operon, even if lactose is present in the culture medium

DNA methylation

-method by which cells can regulate transcription initiation -a biochemical modification of DNA itself in which a methyl group is added to the 5th carbon of the cytosine base in a 5' CpG 3' dinucleotide pair on one strand of the double helix (p stands for phosphate) -enzymes called DNA methyl transferases (DNMTs) catalyze the methylation of cytosines in CpG dinucleotides -DNA methylation can alter gene expression heritably without changing the base sequence of DNA, and thus constitutes an epigenetic phenomenon

initiation of transcription as the first step in gene expression and regulation in prokaryotes

-one of the fundamental modes for regulating the expression of many genes involves the binding of regulatory proteins to DNA targets at or near promoters to control transcription -the DNA binding of these regulatory proteins either inhibits or enhances the effectiveness of RNA polymerase in initiating transcription -we consider the inhibition of RNA polymerase activity as "negative regulation" and the enhancement of RNA polymerase activity as "positive regulation"

trp RNA leader folding into terminator

-one stable conformation of the trp RNA leader contains two stem-loop structures: region 1 makes a stem loop with region 2, while region 3 associates with region 4. the 3-4 stem loop configuration is called a terminator bc when it forms in the trp operon transcript, RNA polymerase contacts it and stops transcription, producing a short "attenuated" RNA

E. coli's utilization of Lactose as a model system of gene regulation

-proliferating e coli can use any of several sugars as a source of carbon and energy. one of these is lactose, a complex sugar composed of two monosaccharides: glucose and galactose -a membrane protein, Lac permease, transports lactose in the medium into the E. coli cell. there, the enzyme B-galactosidase splits the lactose into galactose and glucose. note that this is a catabolic pathway that breaks down lactose into simpler subcomponents

antisense RNAs

-some bacterial genes are regulated by RNAs that are complementary in sequence to the mRNA bc their transcription template is the opposite strand of DNA -these regulatory RNAs are called antisense RNAs; the mRNAs they regulate are sense RNAs -antisense RNAs range in size from10-1000 nt, and may be complementary to the entire mRNA encoded on the opposite DNA strand, or they may overlap only a part of it -some inhibit translation by base pairing with the sense mRNA and blocking the ribosome binding site -sometimes the double stranded RNA formed by base pairing between the sense mRNA and antisense RNA can be degraded by robonucleases. -also, sometimes antisense transcription can interfere with initiation of transcription of the sense gene

overlap of operator and promoter

-some of the nucleotides in the lac operon operator are also part of the lac operon promoter -means that if the operator site is occupied by the repressor (as would happen in a normal cell in the absence of lactose), RNA polymerase cannot "see" the promoter nor bind to it

studies of merodiploids (partial diploids)

-studies in which a second copy of the lac genes was introduced to help distinguish mutations in the operator site (o^c), which act in cis, from mutations in lacI, which encodes a protein that acts in trans. -the merodiploids were made using F' plasmids that carry a few chromosomal bacterial genes. when F' (lac) plasmids are present in a bacterium, the cell has two copies of the region containing both the lactose-utilization genes and lacI-- one on the plasmid and one on the bacterial chromosome. -using F' (lac) plasmids, Monod's group created bacterial strains with diverse combinations of regulatory (o^c and lacI) mutations and mutations in enzyme-encoding structural genes (lacZ and lacY). -the phenotypes of these partially diploid cells allowed the researchers to determine whether particular constitutive mutations were in the genes that produce diffusible, trans-acting proteins or at cis-acting DNA sites that affect only genes on the same molecule.

basal factors; activators and repressors

-the binding of proteins to a genes promoter and enhancer controls the rate of transcriptional initiation -basal factors bind to the promoter. they assist the binding of RNA pol II to the promoter. key component of the complex is the TATA box-binding protein, or TBP. this protein recruits other proteins called TBP-associated factors, or TAFs, to the promoter in an ordered pathway of assembly. once the basal factor complex has formed, RNA pol can initiate a low level of transcription -activators and repressors bind to the enhancers

determining which of the two alternative RNA structures form

-the early translation of a short portion of the RNA leader (while transcription of the rest of the leader is still taking place) determines which of the two alternative RNA structures form -that key portion of the RNA leader includes a short open reading frame containing 14 codons, two of which are trp codons. -when tryptophan is present, the ribosome moves quickly past the trp codons in the RNA leader and proceeds to the end of the leader's codons, allowing formation of the terminator. -in the absence of tryptophan, the ribosome stalls at the two trp codons in the RNA leader bc of the lack of charged tRNA^Trp in the cell. the antiterminator is then able to form, which prevents formation of the terminator. as a result, transcription proceeds through the leader into the structural genes. -the attenuation mechanism allows the cell to avoid expending energy synthesizing gene products unnecessarily; the cell "reads" the level of charged tRNA^Trp and adjusts the level of trp mRNA accordingly

rotational symmetry of the operator

-the lac operator sequences display the interesting property of rotational symmetry. their two DNA strands have an almost identical sequence when read in the 5' to 3' direction on both strands. -this symmetry makes sense when you consider that one form of the Lac repressor protein is a dimer. one of the subunits of the dimer forms tight contacts with the bases making up one-half of the operator sequence. the other subunit of the Lac repressor dimer faces in the opposite direction, and it associates with the other, rotationally symmetric half of the operator sequence.

eukaryotic corepressors

-the primary function of repressors in eukaryotes is to recruit corepressor proteins to enhancers. can have two functions: 1. some corepressors can interact directly with the RNA pol II basal complex and prevent it from binding to the promoter 2. other corepressors are enzymes that modify histone tail amino acids, resulting in closed chromatin. such enzymes are histone deacetylases (HDACs) and histone methyl transferases (HMTs)

Domains of the Lac repressor protein

-the purified repressor protein is a dimer of two identical lacI-encoded subunits; in some situations, two dimers of Lac repressor can associate to form a tetramer. -each subunit contains three distinct domains: 1. one region to bind to the inducer 2. second domain recognizes and binds to DNA at operator sites. 3. third domain is found at the C-terminal region of the polypeptide and interacts with the same domain of other subunits to allow formation of the dimeric and tetrameric proteins -lacI- mutations encoding proteins that could not bind to the operator affected amino acids in the protein's DNA-binding domain. -lac^s superrepressor mutations, encoding proteins that could not be induced, were clustered in codons for amino acids in the inducer-binding domain

induction of coordinated gene expression by lactose

-the two proteins Lac permease and B-galactosidase, both required for lactose utilization, are present at very low levels in cells grown without lactose. the cell has no need for either of these proteins if lactose is not present. the addition of lactose to the bacterial medium causes a 1000-fold increase in the production of these proteins. -the process by which a specific molecule stimulates synthesis of a given protein is known as induction. the molecule responsible for stimulating production of the protein is called the inducer. -in the regulatory system under consideration, lactose modified to a derivative known as allolactase is the inducer of the genes for lactose utilization

lac operon

-three structural genes (lacZ, lacY, lacA) encoding proteins needed for lactose utilizationn, together with two regulatory elements-- the promoter (P) and the operator (o)-- make up the lac operon: a single DNA unit enabling the simultaneous regulation of the three structural genes in response to environmental changes. -molecules that interact with the operon include the repressor, which binds to the operon's operator, and the inducer (allolactose), which when present, binds to the repressor and prevents it from binding to the operator. -proteins bind to DNA to regulate gene expression; this holds true for both the positive and negative regulation of the lac operon

mediator

-transcription of many eukaryoteic genes requires a multi-subunit complex called Mediator that contains more than 20 proteins. Mediator does not bind DNA directly but instead serves as a bridge between the RNA pol II complex at the promoter, and activator or repressor proteins bound at the enhancer

cooperativity in binding of the Lac repressor protein

-two dimers of the Lac repressor protein can associate to form a tetramer -the Lac protein can actually bind to 3 sites in the vicinity of the operon. one of these sites is called o1. the other sites are o2 and o3. -site o1 has the strongest affinity for the repressor, and one of the dimers making up the tetramer always binds to the rotationally symmetrical sequences at this site. -the other dimer within the tetramer binds to either o2 or o3. mutations in either o2 or o3 thus have very little effect on repression. by contrast, mutations in both o2 and o3 make repression 50 times less effective. the conclusion is that for maximum repression, two dimers (and thus all four of the tetrameric repressor's subunits) must bind DNA simultaneously. -the o1 site is roughly 400 bases away from o2 and 100 bases away from o3. these distances are sufficiently large so that a tetrameric repressor molecule can bind simultaneously to o1 and either 02 or o3 only if a loop of DNA forms between operator sites. binding at 4 recognition sequences (2 in each of two operator sites) increases the stability of the protein-DNA interactions so much that it can compensate for any energy required to form the loop. in fact, the DNA binding of the Lac repressor is so efficient that only 10 repressor tetramers per cell are sufficient to maintain repression in the absence of lactose

what activators do

1. activators help 'recruit' the basal factors and pol II to core promoter sequences by interacting directly with the components of this complex 2. activators recruit coactivators; these are proteins that open local chromatin structure to allow gene transcription -HAT enzymes and chromatin remodeling complexes are coactivators that open chromatin

regulating the lactose-utilization genes: induction

1. when lactose is present, allolactose, an inducer derived from the sugar, binds to the repressor. this binding changes the shape of the repressor, making it unable to bind to the operator 2. with the release of the repressor from the operator, RNA polymerase gains access to the lac operon promoter and initiates transcription of the three lactose-utilization genes into a single polycistrinic mRNA -during translation, lacZ becomes B-galactosidase; lacY becomes permease; lacA becomes transacetylase

insulators

DNA elements called insulators organize chromatin so that enhancers have have access only to particular promoters -insulators bind a protein called CTCF that facilitates the formation of DNA loops. a promoter and enhancer will be in separate loops and cannot interact with each other if the insulator is between them

CpG islands

DNA sequences that may be a few 100 or a few 1000 bp long, and within which the frequency of CpG dinucleotides is much higher than that of the rest of the genome -the C residues in CpG islands are usually unmethylated. when the islands in the vicinity of a genes promoter are unmethylated, the chromatin is "open" and the gene is transcriptionally active. methylation of the CpG islands "closes" the chromatin and represses transcription

sRNAs

bacterial genomes encode many small RNA molecules, or sRNAs, that regulate transcription in trans by base pairing with mRNAs -regulatory sRNAs are typically 50-400 nt long, and their sequences contain a region complementary to several different mRNA targets. -most sRNAs are repressive, meaning that they inhibit translation of their target mRNAs by base pairing with the ribosome binding site -some sRNAs activate translation of their target mRNAs by disrupting the formation of a stem loop structure in the leader of the mRNA that would otherwise block the ribosome binding site. -they can also promote the degradation of the mRNA: the double stranded RNA region resulting from sRNA binding to the mRNA causes the mRNA to be degraded by ribonuclease enzymes -sRNA usually do not respond to environmental changes directly. instead, the transcription or stability of an sRNA is often controlled through other regulatory molecules, such as transcription factors that interact with the sRNA gene promoter so as to increase or decrease the cellular concentration of the sRNA -therefore, sRNAs often act as intermediaries in regulatory cascades in which one regulator influences the expression of a different regulator

operons

because some genes form a cluster, they can be transcribed together into a single mRNA, and thus anything that regulates the transcription of this mRNA will affect all the genes in the cluster. clusters of genes regulated in this way are called operons.

elements acting in trans

can diffuse through the cytoplasm and act as target DNA sites on any DNA molecule in the cell

elements acting in cis

can influence only the expression of adjacent genes on the same DNA molecule.

regulation of prokaryotic gene expression

can occur at many different levels: transcription initiation, elongation, or termination; mRNA stability; translation initiation; or protein stability or activity

INDUCIBLE REGULATION

catabolic pathways demand inducible regulation: this means the pathway should be turned on- that is, induced- only when the complex molecules to be broken down (catabolites) are present in the cell's environment. the cell would waste resources synthesizing the enzymes needed to break down a particular sugar if that sugar was not available to the cell.

eukaryotic gene expression

control of eukaryotic gene expression occurs at many levels; these include transcription initiation, transcript processing, mRNA stability, mRNA translation, protein modifications, and protein stability

PaJaMo experiment

further evidence that lacI encodes this hypothetical negative regulator of the lac genes: -researchers transferred the lacI+ and lacZ+ alleles into a bacterial cell devoid of LacI and LacZ proteins in a medium containing no lactose. shortly after the transfer of the lacI+ and lacZ+ genes, the researchers detected synthesis of B-galactosidase. within an hour, however, synthesis stopped. -results were interpreted as follows: when the donor DNA is first transferred to the recipient, there is no repressor (LacI protein) in the recipient cell's cytoplasm bc the recipient cell's chromosome is lacI-. in the absence of repressor, the lacY and lacZ genes are expressed. over time, the recipient cell begins to make the Lac repressor protein from the lacI+ gene introduced by the mating, so expression is again repressed. -Monod and co. proposed that the repressor protein prevents further transcription of lacY and lacZ by binding to a hypothetical operator site: a DNA sequence near the promoter of the lactose-utilization genes -the binding of repressor to this operator site blocks the promoter, and this binding occurs only when lactose is not present in the medium. -also predicted that although some lacI- alleles would be null mutations that could not make any protein, other lacI- mutations would make a form of the repressor protein that was unable to bind to the operator -in final step of the experiment, the researchers added lactose- the precursor of the inducer- to the culture medium. with this addition, the synthesis of B-galactosidase resumed. interpretation of this result was that the inducer binds to the wild-type repressor, changing the shape of the repressor protein so that it can no longer bind to DNA. when the inducer is removed from the environment, the repressor, free of inducer, reverts to its DNA-bindable shape. -the binding of inducer to repressor thus causes an allosteric effect that abolishes the repressor's ability to bind the operator. the inducer is an effector molecule that releases repression without itself binding to the DNA.

regulating the lactose-utilization genes: repression

in the absence of lactose, the repressor binds to the DNA of the operator, and this binding prevents transcription. the repressor thus serves as a negative regulatory element.

superrepressor (lacI^s) mutations

lacI^s mutants, although they cannot bind inducer, can still bind to DNA and repress transcription of the operon. this repressed state is independent of the presence or absence of lactose or allolactose. -such mutations result in cells that cannot turn on the operon, even when inducer was added to the medium.

key lac operon phenotypes: I- o+ Z+ Y+

lacZ activity: w/o inducer (+); w inducer (+) lacY activity: w/o inducer (+); w inducer (+) -conclusion: I encodes repressor. inactive I means no functioning repressor, so lacZ and lacY can be produced without an inducer

key lac operon phenotypes: I+ o^c Z+ Y+

lacZ activity: w/o inducer (+); w inducer (+) lacY activity: w/o inducer (+); w inducer (+) -conclusion: o+ is a DNA site that binds repressor

key lac operon phenotypes: I+ o^c Z+ Y-/F' (I+ o+ Z- Y+)

lacZ activity: w/o inducer (+); w inducer (+) lacY activity: w/o inducer (-); w inducer (+) conclusion: o+ and o^c act in cis

key lac operon phenotypes: I+ o+ Z+ Y+

lacZ activity: w/o inducer (-); w inducer (+) lacY activity: w/o inducer (-); w inducer (+) -lac operon is inducible

key lac operon phenotypes: I- o+ Z+ Y-/F' (I+ o+ Z- Y+)

lacZ activity: w/o inducer (-); w inducer (+) lacY activity: w/o inducer (-); w inducer (+) conclusion: I+ is dominant to I-; repressor acts in trans and is therefore able to diffuse inside the cell and bind to any operator site it encounters, regardless of the operator's chromosomal location

key lac operon phenotypes: I^s o+ Z+ Y+

lacZ activity: w/o inducer (-); w inducer (-) lacY activity: w/o inducer (-); w inducer (-) -conclusion: I^s encodes superrepressor

key lac operon phenotypes: I+ o+ Z+ Y-/F' (I^s o+ Z- Y+)

lacZ activity: w/o inducer (-); w inducer (-) lacY activity: w/o inducer (-); w inducer (-) conclusion: I^s is dominant to I+; superrepressor acts in trans

epigenetic changes

modifications to genes that alter gene expression without changing the base pair sequence and that are inherited directly through cell divisions

constitutive mutants

mutations in a gene called lacI, located near but not within the lac operon, produce constitutive mutants that synthesize B-galactosidase and Lac permease even in the absence of lactose. -constitutive mutants synthesize certain enzymes all the time, irrespective of environmental conditions.

anabolic pathways

other metabolic pathways in the cells allow cells to construct end product molecules they need, such as amino acids and nucleotides, from simpler constituents.

prokaryotic gene regulation

prokaryotes regulate gene expression by activating, increasing, diminishing, or preventing the transcription of specific genes and/or translation of the mRNAs made from these genes. -to adapt and survive in a constantly changing world, bacteria must be able to tune the expression of many genes in a coordinated way so that the cells respond appropriately to many different environments and do not waste energy by making unneeded proteins

TATA box

promoters are an essential DNA sequence in eukaryotic genomes. promoters are always very close to the genes protein-coding region and usually contain a TATA box (or initiation box) consisting of roughly 7 nucleotides of the sequence TATA(A or T)A(A or T), located just upstream of the transcription initiation site. -binding of RNA polymerase to the TATA box allows a low, so called basal level of transcription

operon theory

proposed model of gene regulation which suggested that a single signal can simultaneously regulate the expression of several genes that are clustered together on a chromosome and are involved in the same process.

silencing

repression of genes by DNA methylation is often long-term bc the methylation pattern is maintained through numerous cell divisions; long-term repression through DNA methylation is called silencing

coordinate gene expression

the clustering of genes with similar functions into operons is a simple and efficient way to achieve coordinate gene expression. it is thus not surprising that many operons have evolved in bacterial genomes. -with a cluster of genes, mutations in the promoter affect the transcription of all the genes in the cluster -for each of the genes to be translated from this polycistronic mRNA in bacterial cells, each open reading frame must be preceded by its own independent ribosome binding site

transcription factors

term to describe all sequence-specific DNA binding proteins that influence transcription. -trans acting proteins that include basal factors and activators and repressors -once transcription factors bind to the DNA, they recruit additional proteins to the gene that can also influence transcription

trp RNA leader folding into antiterminator

the alternative RNA structure, called the antiterminator, forms by base pairing between regions 2 and 3. in this conformation, the leader RNA cannot form the terminator (bc region 3 is no longer available to pair with region 4), and as a result, the transcription machinery continues to produce a full-length mRNA that includes the trp structural gene sequences

structural components of the lactose operon in E. coli

the coordination of various elements enables bacteria to utilize lactose in an energy-efficient way. these elements include: 1. a closely linked cluster of three structural genes--lacZ, lacY, lacA-- that encode the enzymes active in splitting lactose into glucose and galactose 2. a promoter site, from which RNA polymerase initiates transcription of a polycistronic mRNA. the promoter acts in cis, affecting the expression of only downstream structural lac genes in the same DNA molecule 3. a cis-acting DNA operator site lying very near the lac operon promoter on the same DNA molecule. the three structural genes together with the promoter and operator constitute the lac operon 4. a trans-acting repressor that can bind to the operator. the repressor is encoded by the lacl gene, which is separate from the operon and is unregulated. after synthesis, the repressor diffuses through the cytoplasm and binds to its target 5. an inducer that prevents the repressor's binding to the operator. the inducer is allolactase, a molecule derived from and thus related to lactose.

repressor

the existence of constitutive mutants suggests that lacI encodes a negative regulator, or repressor. cells would need such a repressor to prevent expression of lacY and lacZ in the absence of an inducer. in constitutive mutants, however, a mutation in the lacI gene generates a defect in the repressor protein that prevents it from carrying out this negative regulatory function.

why gene regulation is more complex in eukaryotes than prokaryotes

the mechanisms of gene regulation in eukaryotes are more complex than those in prokaryotes bc eukaryotes have chromatin, eukaryotic transcripts require more processing, and transcripts are exported from the nucleus to the cytoplasm for translation. in multicellular eukaryotes, complex gene regulation directs development of numerous cell types

catabolite repression

the overall effect of glucose in preventing lac gene transcription is known as catabolite repression, bc the presence of a preferred catabolite (glucose) represses transcription of the operon

genomic imprinting

the unusual phenomenon in which the expression of an allele depends on the parent that transmits it is known as genomic imprinting. in it, the copy of a gene an individual inherits from one parent is transcriptionally inactive, while the copy inherited from the other parent is active. -imprinting results from sex-specific DNA methylation of cis-acting elements (called imprinting control regions (ICR)) that control the expression of particular genes. can silence the maternal or paternal copy of an imprinted gene -during meiosis, the old imprints are erased and new sex-specific methylation patterns are established. -the expression patterns of about 100 human genes depend on whether they were inherited from the male or female parent. paternally imprinted genes are silenced when inherited from the father, while maternally imprinted genes are silenced when inherited from the mother

operator mutants

when mutations change the nucleotide sequence of the operator, the repressor is unable to recognize and bind to the site; the resulting phenotype is the constitutive synthesis of the lactose-utilization proteins. -researchers have isolated constitutive mutants whose genetic defects map to the lac operator site, which is adjacent to the lacZ and lacY genes. they call the constitutive operator DNA alterations o^c mutations.