Chapter 14- GENETICS

lactose permease

allows the uptake of lactose into the bacterial cytoplasm. It cotransports lactose with H+.

oeprator site

also known as the operator is a sequence of bases that provides a binding site for a repressor protein.

AraC

encodes a genetic regulatory protein called the AraC protein. This protein can bind to three different regulatory sites called the araI, araO1 and araO2. Pc is the promoter for the araC gene.

trp operon

encodes enzymes that are needed for the biosynthesis of the amino acid tryptophan. trpE, trpD, trpC, trpB, and trpA genes encode enzymes involved in typtophan biosynthesis.

What can riboswitches regulate

transcription, translation, RNA stability, and splicing.

Inducible operons encode....while repressible operons encode...

Inducible operons encode catabolic enzymes and repressible operons usually encode anabolic enzymes. The lac and ara operon are inducible systems regulated by sugar molecules, while the the trp operon is repressible operon regulated by trp.

a TPP riboswitch in E.col that regulates translation

When TPP levels are low the 5' end of the mRNA folds into a structure that contains a stem loop called the SD anti-sequestor. When this stem loops forms the SD is available for ribosome binding. Therefore the mRNA is translated when TPP is in short supply. When TPP levels are high TPP binds to the RNa and causes a change in the secondary structure. A stem loop forms that contains the SD and the start codon. The formation of this stem loop sequesters the SD sequence, thereby preventing ribosomal binding. This blocks the translation of the enzymes that are needed to make more TPP.

antisense RNA

an RNA strand that is complementary to a strand of mRNA.

polycistronic RNA

an RNA that contains the sequences for two or more genes.

the 5' end of the thiMD mRNA of E.coli

can exist in two different secondary structures that greatly resemble those that occur at the 5' end of the thi operon of B. subtilis.

lac operon

contains a CAP site, promoter (lacP), operator site (lacO), three structural genes (lacZ, lacY and laxA), and a terminator.

high trp levels, 3-4 stem loop forms

coupled transcription and translation occur under conditions in which a sufficient amount of trp is present in the cell. Translation of the trpL gene progresses to it stop codon where the ribosome pauses. This blocks regions 2 from H bonding with any region and thereby enables region 3 to H bond with region 4. This terminates transcription at the U rich attenuator.

lacA

encodes galactoside transacetylase an enzyme that covalently modifies lactose and lactose analogs. This could prevent toxic build up in bacterial cytoplasm.

regulation in transcription

genetic regulatory proteins bind to the DNA and control the rate of transcription. In attenuation transcription terminates soon after it has begun due to the formation of a transcriptional terminator.

allolactose and arabinose act as

inducers in the lac and ara operon respectively.

When allolactose is available

it binds to the repressor. This alters the configuration of the repressor protein that prevents it from binding to the operator site. Therefore RNA polymerase can transcribe the operon.

What are the two transcriptional units present in lactose utilization

lac operon and the lacI gene.

glucose and no lactose

low cAMP, transcription is very low due to the lack of CAP binding and by the bindings of the repressor.

both lactose and gluocse

low caMP, transcription is low due to the lack of CAP binding.

Why do operons occur in bacteria

one biological advantage of an operon organization is that is allows a bacterium to coordinately regulate a group of two or more genes that are involved with a common functional goal.

translational regulatory protein

recognizes sequences within the mRNA much as transcription factors recognize DNA sequences. In most cases they act to inhibit translation, which are known as translational repressors.

micF

responsible for inhibiting the expression of the ompF gene at high osmolarity.

lacP

responsible for the transcription of lacZ, lacY, and lacA gene as a single unit which ends at the lac terminator.

i promoter

responsible for the transcription of the lacI gene.

CAP site

short DNA segments that function in gene regulaton. IT is a DNA sequence recognized by an activator protein called the catabolite activator protein ( CAP).

allosteric proteins have two binding sites

the effector molecule binds to the proteins allosteric site which is a site other than the proteins active site.

In the absence of the inducer allolactose..

the repressor protein ins tightly bound to the operator site thereby inhibiting the ability of RNA polymerase to transcribe the operon.

difference between cis and trans effects

the trans effet is mediated by genes that encode regulatory protein whereas a cis effect is mediated by DNA sequences that are bound by regulatory proteins.

no lactose or glucose

there is high caMP, transcription however is low because there is the binding of the repressor.

gene regulation in translation

translational repressor proteins can bind to the mRNa and prevent translation from starting. Riboswitches can produce an RNA conformation that prevents translation from starting. And antisense RNA can bind to the mRNA and prevent translation from starting.

constitutive genes

unregulated genes. They encode proteins that are continuously needed for the survival of the bacterium. They have essentially constant levels of expression.

post translational covalent modification

Certain types of modifications are involved primarily in the assembly and construction of a functional proteins. They include: proteolytic processing: disulfide bond formations, and the attachment of prosthetic groups, sugars, or lipids. They are typically irreversible changes required to produce a functional product. However phophorlyation, acetylation, and methylation are often reversibile modiciation that transiently affect the function of a protein.

trpL gene

It encodes a peptide containing 14 amino acids called the leader peptide.

Genetic regulatory proteins that respond to small effector molecules have two functional domains.

One domain is a site where the protein binds to the DNA and the other domain is the binding site for the effector molecule.

Activator protein, inducer molecules, and inducbile gene

The activator protein cannot bind to the DNA unless an inducer is present. When the inducer is bound to the activator protein this enables the activator protein to bind to DNA and activate transcription.

activator protein, inhibitor molecules, and repressible gene

The activator protein will bind to the DNA without the aid of an effector molecule. The presence of a inhibitor causes a conformational change that release the activator protein from the DNA. This inhibts transcription.

Benefits of gene regulation

The encoded proteins are produced only when they are required. Therefore the cell avoids wasting valuable energy making proteins it does not need.

What two features are key in the attenuation mechanism

Twp trp codons are found within the mRNA that encodes the trp leader peptide. Also the mRNa can form stem loop structures. The type of stem loop structure that forms underlies attenuation.

diauxic growth

caused by the presence of two sugars, one of which is easier for the target bacterium to metabolize. An example is when both lactose and glucose are present the glucose is metabolized first and then a second sugar is metabolized after glucose is depleted from the environment.

Response to enviornmental stress

certain proteins help a bacterium to surviv enviornmental stress such as osmotic shock or heat shock. These proteins are required only when the bacterium is confronted with the stress.

lacZ

encodes the enzyme B-galactosidase, an enzyme that cleaves lactose into galactose and glucose. As a side B galactosidase also converts a small percentage of lactose into allolactose. Allolactose acts as a small effector molecule to regulate the lac operon. It can be broken down into lactose and glucose.

cis acting factor

is a DNA segment that must be adjacent to the gene(s) that it regulates and is said to have a cis effect on the gene expression. Ex. lac operator site

repressor

is a regulatory protein that binds to the DNA and inhibits transcription. It is termed negative control.

lacI gene

not in the lac operon it is a transcriptional unit involved in genetic regulation in the lacI gene which is constitutively expressed at fairly low levels, has its own promoter, the i promoter. This gene encodes the lac repressor.

Corepressor/Inhibitor

A corepressor is a small molecule that binds to a repressor protein thereby causing the protein to bind to the DNA. An inhibitor binds to an activator protein and prevents it from binding to DNA. And therefore these gens are called repressible genes.

Two types of regulatory proteins are common

A repressor and a activator

What is the trp operons regulated by

A repressor protein and also by attenuation.

cyclic AMP (cAMP)

Glucose does not directly binds to the genetic regulatory protein. Instead it is the small effector molecule cAMP. It is produced from ATP via an enzyme known as adenylyl cyclase. When a bacterium is exposed to glucose the transport of glucose into the cell stimulates a signaling pathway that causes the intracellular concentration of cAMP to decrease because the pathway inhibits adeylyl cyclase enzyme needed for cAMP synthesis. It is an inducer.

repressor protein, corepressor molecules, and repressible gene

In the absence of a corepressor this repressor protein will not bind to the DNA. Therefore transcription can occur. When the corepressor is bound to the repressor protein, this causes a conformational change that allows the protein to bind to the DNA and inhibit transcription.

Repressor protein, inducer molecule, and inducible gene

In the absence of the inducer this repressor protein blocks transcription. The presence of the inducer causes a conformational change that inhibits the ability of the repressor protein to bind to the DNA. Transcription proceeds.

lacI-

It is very close to the lac operon. It is a type of mutant and it is results in the constitutive expression of the lac operon even in the absence of lactose. the correct hypothesis is that the lacI- mutation eliminates the function of the lac repressor. The incorrect hypothesis is that the lacI -mutation result sint eh synthesis of an internal inducer.

Metabolism

Some proteins function in the metabolism of small molecules. For ex. certain enzymes are needed for a bacterium to metabolize particular sugars. These enzymes are required only when the bacterium is exposed to such sugars in its environment.

Why is catabolite repression an advantage?

The explanation is efficiency. The bacterium does not have to express all of the genes necessary for both glucose and lactose metabolism. If glucose is all used up catabolite repression is alleviated and the bacterium expresses the lac operon.

How were the operator site results interpreted

The lac repressor must bind to 01 and either 02 or 03 to cause full repression. As you can see the operator sites are far form each other and for this reason is was proposed that the binding of the lac repressor to two operator sites requires the DNA to form a loop. A loop in the DNA would bring the operator sites closer together thereby facilitating the binding of the repressor protein. The loop is formed between the o1 and o2 or the o1 and o3.

me

The two lacI genes in a merozygote may be different alleles. The lacI - on the chromosome and the lacI+ on the F+ factor. Also the genes on the F' factor and the genes on the bacterial chromosome are not physically adjacent to each other.

The process of a TPP riboswitch in B. subtilis that regulates transcription.

When there is low TPP, and TPP is the active form of thiamin/viatmin b1, which is called thiamin pyrophaspate, When TPP levels are low the secondary structure has a stem loop called an antiterminator which prevents the formation of the terminator stem loop. And therefore under these conditions transcription of the entire thi operon occurs. In this way the bacterium is able to make more TPP which is in short supply. When TPP levels are high, TPP binds to the RNA and causes a change in its secondary structure. a p independent terminator forms instead of the aniterminator stem loop. And the p independent terminator abruptly stops transcription and thereby inhibiting the production of the enzymes that are needed to make more TPP. This is an example like trp operon of attenuation.

The binding of the cAMP-CAP complex to the DNA causes

a 90 degrees bend in the DNA structure. When the repressor is active and not bound to allolactose the cAMP-CAP complex facilitates the binding of the lac repressor to the o1 and o3 sites. When the repressor is inactive this bending also appear to be important in the ability of RNA polymerase to initiate transcription slightly downstream form the bend.

feedback inhibition

a common way to regulate the activity of metabolic enzymes .The synthesis of many cellular molecules such as amino acids, vitamin, and nucleotides occurs via the action of a series of enzymes that convert precursor molecules to a particular product. The final product in a metabolic pathway then inhibits an enzyme that acts early in the pathway. if the concentration of the final product becomes high it will bind to the enzyme 1 and inhibit its ability to convert the substrate into intermediate one.

trans effect

a form of genetic regulation that can occur even though the two DNA segments are not physically adjacent. Ex. The action of the lac repressor on the lac operon is a trans effect.

operon

a group of two or more genes under the transcriptional control of a single promoter. It encodes a polycistronic RNA

riboswitches

a method of gene regulation. In this form of regulation an RNA molecule can exist in two different secondary conformations. The conversion of one conformation to another is due to the binding of a small molecule. They are widespread in bacteria.

Inducer

a small effector molecule that causes transcription to increase. An inducer may accomplish this in two ways: it could bind to a repressor protein and prevent it from binding to DNA or it could bind to an activator protein and cause it to bind to DNA. In either case transcription is increased. Genes regulated in this manner are called inducible genes.

catabolite

a substance that is broken down inside the cell.

low trp levels, 2-3 stem loop forms

coupled transcription and translation occur under conditions in which the trp concentration is very low. The ribosome pauses at the Trp codons in the trpL gene bc insufficient amount sof charged tRNA (trp) are present. This pause blcoks region 1 of mRNA so region 2 can only bind to region 3. And since no 3-4 stem loop transcriptional termination cannot occur and RNA polymerase transcribes the rest of the operon.

gene regulation in posttranslation

in feedback inhibition the product of a metabolic pathway inhibits the first enzyme in the pathway. Covalent modifications to the structure of a protein can alter its function.

trpL and trpR

involved in regulating the trp operon in two different ways. the trpR gene encodes the trp repressor protein. When trp levels are low the trp repressor cannot bind to the operator site. Under these conditions RNA polymerase transcribes the trp operon. In this way the cell expresses the genes required for the synthesis of tryptophan. When the trp levels become high in the cell trp acts as a corepressor that binds to the rep repssor proteins. This causes a configurational change in the trp repressor that allows it to bind to the trp operator site. This inhibits the ability of RNA polymerase to transcribe the operon. And therefore when the trp is high in the cell the trp operon is turned off because it does not need anymore.

When a translational repressor protein binds to mRNA, it can inhibit translational initiation in one of two ways

it can bind in the vicinity of the SD sequence and or start codon and thereby sterically block the ribosome's ability to initiate translation in this region. Or the repressor protein may bind outside the SD/start codon region but stabilize the mRNA secondary structure that prevents initiation.

posttranslational

regulation refers to the functional control of proteins that are already present in the cell rather than regulation of transcription or translation. Post translational control can either activate or inhibit the function of a protein. When compared to transcription and translation it can be fast.

In the absence of arabinose

the AraC protein binds to the araI , araO1, and araO2 sites. An AraC protein dimer is bound to araO1 whereas monomers are bound at araO2 and araI. The binding of the AraC dimer to the araO1 site inhibits the transcription of the araC gene. And this keeps araC protein levels fairly low. The AraC proteins bound at araO2 and araI can bind to each other by causing a loop in the DNA. This DNA loop prevents RNA polymerase from binding to the DNa and transcribing the ara operon via P(BAD), and therefore in the absence of arabinose the ara operon is turned off.

CAP

the effects of cAMP on the lac operon is mediated by an activator protein called the catabolite activator protein, CAP. It is composed of two subunits, each of whcih binds one molecule to cAMP. It is an activator.

How does the inhibition occur

the micF RNA is complementary to ompF gene at high osmolarity, it is the antisense strand of RNA. When the micF gene is transcribed its RNa product binds to the ompF mRNA via hydrogen bonding between the complementary regions. the binding of the micF RNa to the ompF mRNa prevents the ompF mRNA from translating. The RNA transcribe from the micF gene is called the antisense RNA bc it is complementary to the ompF mRNa which is a sense strand of mRNA that encodes a protein. the micF RNa does not encode a protein.

What is the role of the trp codons

these two codons provide a way to sense whether or not the bacterium has sufficient trp to synthesize its proteins.

small effector molecules

they do not bind directly to the DNA to alter transcription. They exert its effects by binding to an activator or repressor. The binding of an effector molecule causes a conformational change in the regulatory protein and thereby influences whether or not the protein can bind to DNA.

enzyme adaptation

this phenomenon is due to the synthesis of cellular proteins. It refers to the observation that a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme.

The cycle of lac operon induction and repression

1. when lactose becomes available a small amount of it is taken up by lactose permease and converted to allolactose by beta-galactosidase. The allolactose binds to the repressor causing it to fall off the operator site. 2. lac operon proteins are synthesized and this promotes the efficient metabolism of lactose. The binding of the allolactose promotes a confirmational change and prevents the repressor frin binding to the lac operator site and thereby allows transcription of lacY,lacZ, and lacA. 3. the lactose is depleted. Allolactose levels decrease. Allolactose is released from the repressor allowing it to bind to the operator site. 4. Most protein involved with lactose utilization are degraded.

merozygote

A strain of bacteria containing F' factor genes, also called partial diploid.

Allosteric enzyme

An example is enzyme 1, it is an enzyme that contains two binding sites. The catalytic site is responsible for the binding of the substrate and its conversion to intermediate one. The second site it a regulatory or allosteric site. This site binds to the final product of the metabolic pathway. When bound to the regulatory site the final product inhibits the catalytic ability of enzyme one.

How do we explain the above effects

Because the normal lacI gene on the F' factor was not physically located next to the chromosomal lac operon this result is consistent with the idea that the lacI gene codes for a repressor protein that can diffuse throughout the cell and bind to any lac operon. The hypothesis that the lacI- mutation resulted in the synthesis of a internal inducer was rejected. If that hypothesis was correct the merozygote strain would still have an internal inducer and the lac operons in the merozygote would have been expressed in the absence of lactose.

lacY

Encodes lactose permease a membrane protein required for the active transport of lactose into the cytoplasm of the bacterium.

trans acting factor

Ex. A regulatory protein such as the lac repressor.

lacI mutant vs lacO

In the lacI- with no F' factors regardless of lactose present or not there is 100 percent expression of lac operon, this is the same for lacO- When lacI- has f' factors of lacI + and normal lac operson there is 200 percent with lactose and less than 1 percent without it. When lacO- has f' factorsof lacI + and normal lac operon, in the presence of lactose the expression of lac operon is 200% and without lactose it is 100%. As you can see a loss of function mutation in a gene encoding a repressor protein has the same effect as a mutation in an operator site that cannot bind to a repressor protein. in both cases the genes of the lac operon are constitutively expressed. In merozygotes the results are different. When a normal lacI gene and a normal lac operon are introduced into a cell harboring a defective lacI gene the normal lacI gene can regulate both operons. In contrast when a lac operon with a normal operator site is introduced into a cell with a defective operator site the operon with the defective operator site continues to be expressed without lactose present. Overall a mutation in trans acting factor can be complemented by the introduction of a second gene with normal function. However a mutation in the cis acting element is not affected by the introduction of another cis acting element with normal function into the cell.

attenuation

It is mediated by the region that includes the trpL gene. It can occur in bacteria because transcription and translation are coupled. During attenuation transcription actually begins but it is terminated before the entire mRNA is made. A segment of attenuator sequence is important in facilitating terminaiton. When attenuation occurs the mRNA from the trp operon is made as a short piece that terminates shortly past the trpL gene. Bc this short mRNA has been terminated before RNA polymerase has transcribed the trpE trpD trpC trpB and trpA genes it will not encode the proteins required for trp biosynthesis.

ara operson

P(BAD) is the promoter for the ara operon, which contains three genes araB araA and araD, which are involved in encoding proteins in the arabinose mechanism. The three genes encode a polycistronic mRNA for the three enzymes involved in arabinose metabolism. The action of the three enzymes metabolizes arabinose into D-xylulose 5-P arabinose (a) , L-ribulose ( b), l riulose 5-P (d),D xylulose 5-P.

How was attenuation found?

Scientists noticed that mutations in the trp operon still allowed for regulation. As well as mutation sin the trpL gene still allowed for regulation

Cell division

Some proteins are needed for cell division. These are necessary only when the bacterial cell is going to divide.

What observations lead Jacob and Monod that enzyme adaptation is due to the synthesis of specific cellular proteins in response to lactose in the environment.

The exposure of bacterial cells to lactose increase the levels of lactose utilizing enzymes by 100 to 10000 fold, antibody and labeling techniques revealed that the increase in the activity of these enzymes was due to the increased synthesis of the enzymes, the removal of lactose from the environment caused an abrupt termination in the synthesis of the enzymes, and mutations that prevented the synthesis of a particular protein involved in lactose utilization showed that a separate gene encoded each protein.

Lactose, no gluocse.

There is high cAMP, and therefore high rate of transcription bc the binding of RNA polymerase to promoter is enhanced by the CAP binding.

The stem loops in attenuation

These are the stem loop structures that are possible, 1-2, 2-3, 3-4. However if 1 is complementary to 2 and forms a stem loop there then 2 can not form a stem loop with 3, etc.

Bacterial conjugation experiemtn with Monod and Pardee

They applied the genetic approach. They did matings between F- and donor cells which were Hfr strains that transferred a portion of the bacterial chromosome. They found that F' factors that carried the lacI gene and proteins of the lac operon. The f' factors can be transferred from one cell to another by bacterial conjugation.

catabolite repression

This form of regulation is influenced by the presence of glucose, which is a catabolite.The presence of glucose leads to repression of the lac operon. When exposed to both glucose and lactose, E.coli cells first use glucose and catabolite repression prevents the use of lactose.

pseudo operators

This is what O2 and O3 operator sites were called because of substantial repression in the absence of either one of them.

In the presence of arabinose

When arabinose is bound to the AraC protein the interaction between the araC proteins at the araO2 and araI site is broken. This opens the DNA loops. In addition the second AraC protein binds at the araI site. The AraC dimer at the araI operator sites activates transcription by directly interacting with RNA polymerase. This activation can occur in conjugation with the activation of the ara operson by CAP and cAMP if glucose levels are low. When the ara operon is activated the bacterial cell can efficently metabolize arabinose.

lac repressor

a protein that is important for the regulation of the lac operon. The lac repressor functions as a homotetramer a protein composed of four identical subunits. Only a small amount of the lac repressor protein is needed to repress the lac operon.

activator

a regulatory protein that increased the rate of transcription.

osmoregulation

essential for the survival of most bacteria. It refers to the ability to control the amount of water inside the cell. the protein ompF gene is important for osmoregulation. At low osmolarity the ompF protein is preferentially produced whereas at high osmolarity its synthesis is decreased.

lac operon and its operator sites

has three operator sites for the lac repressor. O1, O2, and O3. O1 is the lac operator site and it is slightly downstream form the promoter. O2 is farther dosnwstream in the lacZ coding sequence and O3 is located slightly upstream from the the CAP site. . When all three operator sites are present the repression of the lac operon is 1300 fold. There is 1/1300 the level of expression as compared to when lactose is present. A value of one shown when there are no operator sites indicates that there is constitutive expression. When o2 and 03 is missing and there is only 01 repression is dramatically decreased, and when o1 is missing even in the presence of 02 and 03 repression is nearly abolished.

Conditions that favor the 3-4 stem loop

high trp levels. and when translation is not coupled with transcription. Attenuation occurs when there is a 3-4 stem loop.

Gene regulation

the phenomenon in which the level of gene expression can vary under different conditions.

Evidence that the lacI gene encodes a diffusable repressor protein

the production of beta-galactosidase in the original mutant strain was the same in the presence or absence of lactose and this is because the expression of beta-galactosidase in the lacI- mutant strain was already known to be constitutive. In other words the presence of lactose was not needed to induce the operon due to a defective lacI gene. In the merozygote strain, in the absence of lactose, the lac operon were repressed even the operon on the bacterial chromosome. While the addition of lactose increased the amount of beta galactosidase to 220 %. amount of beta-galactosidase= parental strain.

3-4 stem loop structure

together with the U rich attenuator sequence it acts as an intrinsic terminator. Therefore the 3-4 stem loop causes RNA polymerase to pause, and the U rich sequence dissociates from the DNA. This terminates transcription at the U rich attenuator. In comparison if region 3 forms a stem loop with region 2, transcription will not be terminated because a 3-4 stem loop cannot form.

How can post translational regulation occur

via feedback inhibition and covalent modification

How does a small effector molecule like allolactose exerts its effects

when allolactose binds to the repressor a conformational change occurs in the lac repressor protein that prevents it from binding to the operator site. Therefore the ability of the lac repressor to bind to the operator site depends on whether or not allolactose is bound to it.

when there is no translation

when translation isn't coupled with transcription the most stable for of mRNA is when region 1 hydrogen bonds to region 2. And therefore 3-4. A terminator stem loop forms and transcription will be terminated right past the trpL gene.