Genetics Exam 3

expression of lac operon for lacI- chromosome and no F' factor

100% with lactose and 100% without lactose

expression of lac operon for lacO- chromosome and no F' factor

100% with lactose and 100% without lactose

expression of lac operon for wild type chromosome with no F' factor

100% with lactose and <1% without lactose

when translation is coupled with transcription in low tryptophan levels

2-3 stem loop forms. ribosome pauses at the trp codons in the trpL gene because insufficient tRNA^trp are present and this pause blocks region 1. transcriptional termination does not occur and RNA polymerase transcribes the rest of the operon

expression of lac operon for lacO- chromosome and F' factor with lacI+ and normal lac operon

200% with lactose and 100% without lactose

expression of lac operon for lacI- chromosome and F' factor with lacI+ and normal lac operon

200% with lactose and <1% without lactose

translation riboswitch

5′ end of the mRNA folds into a structure that contains a stem-loop called the Shine-Dalgarno antisequestor. When this stem-loop forms, the Shine-Dalgarno sequence is accessible to ribosome binding. or a stem-loop forms that contains the Shine-Dalgarno sequence and the start codon. The formation of this stem-loop sequesters the Shine-Dalgarno sequence, thereby preventing ribosomal binding.

feedback inhibition

A common mechanism for regulating the activity of metabolic enzymes. a series of enzymes that convert precursor molecules to particular products. The final product in a metabolic pathway then inhibits an enzyme that acts early in the pathway

An operon is a group of two or more genes under the transcriptional control of

A single promoter

merozygote, or partial diploid

A strain of bacteria containing F′ factor genes. production of merozygotes was instrumental in determining the function of the lacI gene

What happens when levels of allolactose rise inside an E. coli cell?

Allolactose binds to the lac repressor and prevents it from binding to the operator.

Repressible Operons Commonly Encode

Anabolic Enzymes. The corepressor or inhibitor is commonly the small molecule that is the product of the enzymes' biosynthetic activities. For example, tryptophan is produced by the sequential action of several enzymes that are encoded by the trp operon. Tryptophan itself acts as a corepressor that binds to trp repressor when the intracellular levels of tryptophan become relatively high. This mechanism turns off the genes required for tryptophan biosynthesis when enough of this amino acid has been made. Therefore, genetic regulation via repression provides the bacterium with a way to prevent the overproduction of the product of a biosynthetic pathway

lac operon contains several DNA sequences:

CAP site; lac promoter (lacP); an operator site (lacO); three protein-encoding genes, lacZ, lacY, and lacA; and a terminator

Inducible Operons Usually Encode

Catabolic Enzymes. The substance to be broken down or a related compound often acts as the inducer. For example, allolactose acts as an inducer of the lac operon. An inducible form of regulation allows the bacterium to express the appropriate genes only when they are needed to break down the sugars or other substances

The somewhat imprecise term that describes how transcriptional regulation is influenced by glucose is

Catabolite repression

Posttranslational Regulation Can Occur via

Feedback Inhibition or Covalent Modifications

some common processes regulated at genetic level

Metabolism: Some proteins function in the metabolism of small molecules. Response to environmental stress: Certain proteins help a bacterium to survive an environmental stress such as osmotic shock or heat shock. Cell division: Some proteins are needed for cell division

lac repressor can bind to

O1 and O2, or to O1 and O3, but not to O2 and O3. If either O2 or O3 is missing. maximal repression is not achieved because it is less likely that the repressor will bind when only two operator sites are present. When O1 is missing, even in the presence of the other operator sites, repression is nearly abolished because lac repressor cannot bind to O2 and O3. the operator sites are a fair distance from each other.

pseudo-operators

O2 and O3. because substantial repression occurred in the absence of either one of them. however, if both O2 and O3 are missing, repression is dramatically reduced even when O1 is present

eukaryotic genes when unregulated are

OFF

RNA stability riboswitch

One mRNA conformation may be stable, whereas the other conformation acts as ribozyme that causes self-degradation.

mRNA made from the trpL gene has four regions that are complementary to each other, which causes the mRNA to form stem-loops. Different combinations of stem-loops are possible due to interactions among these four regions.

Region 2 is complementary to region 1 and also to region 3. Region 3 is complementary to region 2 as well as to region 4. Region can only be engaged in one at a time though. the 3-4 stem-loop is functionally unique. The 3-4 stem-loop in combination with the U-rich attenuator sequence results in intrinsic termination, also called ρ-independent termination which causes RNA polymerase to pause, and the U-rich sequence dissociates from the DNA. This terminates transcription at the U-rich attenuator.

transcription riboswitch

The 5′ region of an mRNA may exist in one conformation that forms a terminator stem-loop, which causes attenuation of transcription. The other conformation does not form such a terminator and is completely transcribed. stem-loop called an antiterminator can prevent the formation of the terminator stem-loop. regulation of TPP biosynthetic enzymes in B. subtilis occurs via a riboswitch that controls transcription

segment of the trp operon immediately downstream from the operator site plays a critical role during attenuation

The first gene in the trp operon is the trpL gene. The mRNA made from the trpL gene contains codons for 14 amino acids that form the trp leader peptide

lacI^S mutations

The lac operon cannot be induced even in the presence of lactose. These mutations (lacI- and lacIs) , which are called super-repressor mutations, alter the region of lac repressor that binds allolactose. The mutation usually results in a lac repressor protein that cannot bind allolactose. If lac repressor is unable to bind allolactose, it will remain bound to the lac operator site and therefore induction cannot occur.

diauxic growth

The sequential use of two sugars by a bacterium. common phenomenon among many bacterial species. Glucose, a more commonly encountered sugar, is metabolized preferentially, and then a second sugar is metabolized after glucose is depleted from the environment

CAP can activate transcription at more than 100 operons in E. coli. Some of these operons encode genes that are needed for the breakdown of sugars.

These include the lac operon. when glucose levels are high, these operons are inhibited. This promotes diauxic growth

cis-acting element

a DNA segment that must be adjacent to the gene(s) that it regulates, and it is said to have a cis-effect on gene expression. The lac operator site is an example of a cis-acting element. A trans-effect is mediated by genes that encode regulatory proteins, whereas a cis-effect is mediated by DNA sequences that are binding sites for regulatory proteins.

binding of the cAMP-CAP complex to the DNA causes

a bend in the DNA structure. When the repressor is active—not bound to allolactose—the cAMP-CAP complex facilitates the formation of a loop in which lac repressor binds to the O1 and O3 sites. When the repressor is inactive, the bending of the DNA also appears to be important in the ability of RNA polymerase to initiate transcription slightly downstream from the bend

operon

a group of two or more genes that are under the transcriptional control of a single promoter. An operon encodes a polycistronic mRNA, an RNA that contains the sequences of two or more genes. the expression of the genes occurs as a single unit. To facilitate transcription, operon is flanked by a promoter that signals beginning of transcription and terminator that signals the end of transcription.

binding of lac repressor to two operator sites requires the DNA to form ________.

a loop. A loop in the DNA brings the operator sites closer together, thereby facilitating the binding of the repressor protein

lacI^-d mutations

a negative dominant allele for a repressor protein that can not bind to DNA and prevents other functional copies of repressor protein from binding to operator site as well

O^c mutations

a nonfunctional set of operators not recognized by repressor protein

trp repressor

a protein encoded by the the trpR gene that regulates the trp operon

activator

a regulatory protein that binds to DNA and increases the rate of transcription. positive control

repressor

a regulatory protein that binds to DNA and inhibits transcription. negative control. has a measurable affinity for allolactose

each protein involved with lactose utilization is encoded by ___

a separate gene

lac repressor binding process to operator sites in lac operon

a tetramer of four identical subunits. The crystal structure revealed that each dimer within the tetramer recognizes one operator site. binding of lac repressor to the O1 and O3 sites. The amino acid side chains in the protein interact directly with bases in the major groove of the DNA double helix. This is how genetic regulatory proteins recognize specific DNA sequences. Because each dimer within the tetramer recognizes a single operator site, the association of two dimers to form a tetramer requires that the two operator sites be close to each other. For this to occur, a loop must form in the DNA. The formation of this loop dramatically inhibits the ability of RNA polymerase to slide past the O1 site and transcribe the operon.

allosteric regulation

action of a small effector molecule, such as allolactose. functioning of allosteric proteins, such as lac repressor, is controlled by effector molecules that bind to the proteins' allosteric sites

When only lactose is present, _______ and ______ levels are high. _______ binds to lac repressor and prevents it from binding to the DNA.

allolactose and cAMP. Allolactose. The effector molecule cAMP binds to CAP, and then CAP binds to the CAP site. A domain in CAP interacts with RNA polymerase, which facilitates the binding of RNA polymerase to the promoter. Under these conditions, transcription proceeds at a high rate

When allolactose is available

allolactose binds to the repressor which alters the conformation of the repressor protein, which prevents it from binding to the operator site and therefore RNA polymerase can transcribe the operon

The binding of allolactose to lac repressor is reversible. The likelihood that allolactose will bind to the repressor depends on the ____________________. During induction of the operon, the concentration of allolactose (the inducer) rises and approaches the affinity for the repressor protein. This makes it likely that allolactose will bind to lac repressor, thereby causing the repressor to be released from the ________.

allolactose concentration, operator site

second way that bacteria regulate translation is via the synthesis of antisense RNA which is

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

trp operon

another operon in E. coli. encodes enzymes involved with the synthesis of the amino acid tryptophan. Like the lac operon, the trp operon is regulated by a repressor protein. also regulated by another mechanism called attenuation, in which transcription begins but is stopped prematurely, or attenuated, before most of the trp operon is transcribed. Attenuation works through a mechanism that depends on coupling of transcription and eukaryotes

inhibitor

binds to an activator protein and prevents it from binding to the DNA. genes they regulate are termed repressible genes

each subunit of lac repressor has a region that _______

binds to the DNA and another region that contains the allolactose-binding site

How does exposing an E. coli cell to glucose affect the regulation of the lac operon via CAP?

cAMP does not bind to CAP and transcription is decreased.

In the absence of both lactose and glucose, _____ levels are also high, so _______ is bound to the DNA.

cAMP. CAP. but the binding of lac repressor is a very strong inhibitor of transcription. For this reason, the transcription rate is very low.

F factors or F′ factors

carries genes that were originally within the bacterial chromosome. carried the lacI gene and the lac operon. These F′ factors can be transferred from one cell to another by bacterial conjugation. F− are recipient cells

The lac operon is transcriptionally regulated in a second way, known as

catabolite repression. This form of transcriptional regulation is influenced by the presence of glucose, which is a catabolite—a substance that is broken down inside the cell. The presence of glucose ultimately 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. If the glucose is used up, catabolite repression is alleviated, and the bacterium then expresses the lac operon

lacA gene

encodes galactoside transacetylase, an enzyme that covalently modifies lactose and lactose analogs by the attachment of hydrophobic acetyl groups. The acetylation of nonmetabolizable lactose analogs prevents their toxic buildup

lacY gene

encodes lactose permease, a membrane protein required for the active transport of lactose into the cytoplasm of the bacterium

lacZ gene

encodes β-galactosidase, an enzyme that cleaves lactose into galactose and glucose. also converts a small percentage of lactose into allolactose, a structurally similar sugar. allolactose acts as a small effector molecule for regulating the lac operon

The research of Monod and Jacob that led to the initial understanding of gene regulation stemmed from an interest in

enzyme adaptation

allosteric enzymes

enzyme w 2 different binding sites. catalytic site is responsible for the binding of the substrate and its conversion to intermediate 1. The second site is a regulatory, or allosteric, site. This site binds the final product of the metabolic pathway. When bound to the regulatory site, the final product inhibits the catalytic ability of enzyme 1. As the final product is made within the cell, its concentration gradually increases. Once the final product concentration has reached a level that is similar to the product's affinity for enzyme 1, the product is likely to bind to the regulatory site on enzyme 1 and inhibit its function. In this way, the accumulation of the final product of a metabolic pathway inhibits the further synthesis of more product.

unregulated genes

essentially constant levels of expression in all conditions over time. constitutive genes

trp operon contains ____________

five genes, designated trpE, trpD, trpC, trpB, and trpA, which encode enzymes involved in tryptophan biosynthesis. In addition, two genes, trpR and trpL, play a role in the regulation of the trp operon. The trpL gene is part of the trp operon, whereas the trpR gene has its own promoter and is not part of the trp operon

trans-effect

form of genetic regulation that can occur even though two DNA segments are not physically adjacent. The action of lac repressor on the lac operon is a trans-effect. A regulatory protein, such as lac repressor, is called a trans-acting factor

posttranslational regulation

functional control of proteins that are already present in the cell rather than the regulation of transcription or translation. Posttranslational regulation can either activate or inhibit the function of a protein. fast process in a matter of seconds. transcriptional and translational regulation typically require several minutes or even hours to take effect because these two mechanisms involve the synthesis and turnover of mRNA and polypeptides

bacterial regulation of gene expression in posttranslation

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

O2 operator site

in the lac operon located farther downstream in the lacZ coding sequence

O3 operator site

in the lac operon located slightly upstream from the promoter.

O1 operator site

in the lac operon slightly downstream from the promoter

In the absence of lactose, no _______ is available to bind to lac repressor. Therefore, lac repressor binds to the ______ and inhibits transcription. does not completely inhibit transcription, so very small amounts of β-galactosidase, lactose permease, and galactoside transacetylase are made but too low to enable the bacterium to readily use lactose. When the bacterium is exposed to lactose, a small amount can be transported into the cytoplasm via lactose permease, and β-galactosidase converts some of that lactose to allolactose. As this occurs, the cytoplasmic level of allolactose gradually rises; eventually, allolactose binds to lac repressor. The binding of allolactose promotes a conformational change that prevents the repressor from binding to the lac operator site, thereby allowing transcription of the ___________ to occur. Translation of the encoded polypeptides produces the proteins needed for lactose uptake and metabolism

inducer (allolactose), operator site, lacZ, lacY, and lacA genes

The lac operon is an _______ operon that is regulated by _______. the trp operon is a _______ operon regulated by _________, a corepressor that binds to the repressor and turns the operon off. In addition, an abundance of charged tRNATrp in the cytoplasm can turn the trp operon off via attenuation.

inducible. sugar molecules. repressible. tryptophan

Genetic regulation of translation is usually aimed at preventing

initiation. riboswitches

CAP site

is a DNA sequence recognized by an activator protein called catabolite activator protein (CAP).

when only glucose is present. The transcription of the lac operon is very low because _______ is bound to the __________ and strongly inhibits transcription

lac repressor. operator site

when both lactose and glucose are present, the presence of lactose causes _______ to be inactive, which prevents it from binding to the _____.

lac repressor. operator site. the presence of glucose decreases cAMP levels, and so cAMP is released from CAP, which prevents CAP from binding to the CAP site. The lack of CAP binding does not completely inhibit transcription. Therefore, the transcription of the lac operon is low in the presence of both sugars.

When the lac repressor binds to the lac operator site RNA polymerase cannot transcribe the

lacZ, lacY or lacA genes

The intracellular concentration of allolactose remains high as long as ______ is available in the environment.

lactose. when lactose is depleted from the environment, the concentration of allolactose also becomes lower due to the action of metabolic enzymes. when the concentration of allolactose drops below its affinity for the repressor, allolactose is unlikely to be bound to lac repressor so lac repressor returns to the conformation that binds to the operator site. the binding of the repressor shuts down the lac operon when lactose is depleted from the environment. After repression occurs, the mRNA and proteins encoded by the lac operon are eventually degraded

riboswitch

mechanism of gene regulation. RNA molecule can exist in two different secondary conformations. The conversion from one conformation to the other is due to the binding of a small molecule. can regulate transcription, translation, RNA stability, or splicing. 3-5% of all bacteria have them

site in the lac operon commonly called the operator site was first identified by

mutations that prevented lac repressor from binding.

gene regulation

phenomenon in which the level of gene expression can vary under different conditions so proteins can be produced at the proper times and in the proper amounts. Saves energy. bacteria exist in an environment that is frequently changing with regard to temperature, nutrients, and many other factors

key feature of attenuation is that two tryptophan (Trp) codons are found within the mRNA. these two codons ______

provide a way for the bacterium to sense whether or not it has sufficient tryptophan to synthesize its proteins. For the ribosome to smoothly progress to the trpL stop codon, enough charged tRNATrp must be available to translate this mRNA. It follows that the bacterium must have a sufficient amount of tryptophan. Under these conditions, the rest of the transcription of the operon is terminated

translational regulatory protein

recognizes sequences within the mRNA, similar to the way that transcription factors recognize DNA sequences. In most cases, translational regulatory proteins act to inhibit translation. These are known as translational repressors

Enzyme adaptation

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. When a bacterium is not exposed to a particular substance, it does not make the enzyme needed to metabolize that substance. removal of substance from environment caused abrupt termination in the synthesis of the enzymes.

when translation is not coupled with transcription

region 1 hydrogen bonds to region 2 and region 3 H bonds to region 4 so stem loop forms and transcription terminated at U-rich attenuator

lac repressor

regulates the lac operon by binding to the operator site and repressing transcription. negative control. This repressor is a homotetramer, a protein composed of four identical subunits (each has a single binding site for allolactose, the inducer.) Only small amount of lac repressor needed to repress the lac operon. Once bound, lac repressor prevents RNA polymerase from transcribing the lacZ, lacY, and lacA genes. The binding of the repressor to the operator site is a reversible process. In the absence of allolactose, lac repressor is bound to the operator site most of the time

bacterial regulation of gene expression in transcription

regulatory proteins bind to DNA and control rate of transcription of one or more nearby genes. in attenuation transcription terminates soon after it begins because of formation of transcriptional terminator

lacI^- mutations

result in the constitutive expression of the lac operon, which means that it is expressed in both the presence and the absence of lactose. Such mutations may result in an inability to synthesize any repressor protein, or they may produce a repressor protein that is unable to bind to the DNA at the lac operator site. If lac repressor is unable to bind to the DNA, the lac operon cannot be repressed.

attenuation

second regulatory mechanism for the trp operon. mediated by the region that includes the trpL gene. During attenuation, transcription actually begins, but it is terminated before the entire mRNA is made. A segment of DNA, termed the attenuator sequence, is important in facilitating this termination. When attenuation occurs, the mRNA from the trp operon is made as a short piece that terminates at the attenuator sequence, which is just downstream from the trpL gene. Because 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 tryptophan biosynthesis. In this way, attenuation inhibits the further production of tryptophan in the cell

posttranslational covalent modification

second strategy to control the function of proteins is the covalent modification of their structure. Certain types of modifications, such as phosphorylation (—PO4), acetylation (—COCH3), and methylation (—CH3), are often reversible modifications that transiently affect the function of a protein.

operator site (also referred to simply as the operator)

sequence of bases that provides a binding site for a repressor protein called lac repressor

cyclic-AMP (cAMP)

small effector molecule involved in diauxic growth. 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 adenylyl cyclase. effect of cAMP on the lac operon is mediated by the activator protein called catabolite activator protein (CAP). CAP is composed of two subunits, each of which binds one molecule of cAMP

corepressor

small effector molecule that binds to a repressor protein, thereby causing the protein to bind to the DNA. genes they regulate are termed repressible genes

inducer

small effector molecule that causes the rate of transcription to increase. may accomplish this in two ways: It can bind to a repressor protein and prevent it from binding to the DNA, or it can bind to an activator protein and cause it to bind to the DNA. Genes that are regulated in this manner are called inducible genes

a repressor protein may bind to the mRNA at a location that is not near the Shine-Dalgarno sequence or the start codon region, but the binding __________

stabilizes an mRNA secondary structure that prevents initiation. a translational repressor could stabilize an mRNA secondary structure in which the Shine-Dalgarno sequence is inaccessible. This would prevent the mRNA from binding to the ribosome, thereby blocking translation.

translational repressor can bind in the vicinity of

the Shine-Dalgarno sequence and/or the start codon thereby sterically blocking the ribosome's ability to initiate translation in this region

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

lacI genes in a merozygote may be different alleles. (this is the repressor gene)

the lacI gene on the chromosome may be a lacI− allele that causes constitutive expression, whereas the lacI gene on the F′ factor may be normal. the presence of lactose is not needed to induce the operon when there's defective lacI gene. β-galactosidase known to be constitutively expressed in the lacI− mutant strain. presence of lactose not needed to induce the operon due to a defective lacI gene but presence of lactose is usually needed the genes on the bacterial chromosome and the genes on the F′ factor are not physically adjacent to each other.

In bacteria, the most common way to regulate gene expression is by influencing

the rate of transcription initiation

a mutation in a ______ factor can be complemented by the introduction of a second gene with normal function. However, a mutation in a ______ is not affected by the introduction of a normal ______ into the cell

trans-acting. cis-acting element, cis-acting element. this is why introduction of normal lacI+ (repressor gene) into chromosome with lacI- gene can result in lac operon not being expressed without lactose. whereas if chromosome has lacO- and F' factor is introduced with lacI+ and normal lac operon, the lac operon will still be expressed in absence of lactose because the lacO- doesn't let repressor bind to it

lacI gene

transcriptional unit. has its own promoter called the i promoter and is constitutively expressed at fairly low levels. The lacI gene encodes lac repressor, a protein that regulates the lac operon by binding to the operator site and repressing transcription

coupling

translation begins as transcription is occurring

when translation is coupled with transcription in high tryptophan levels

translation of trpL gene progresses to its stop codon and ribosome pauses which blocks region 2 from H bonding so 3 and 4 H bond and transcription terminates at U rich attenuator

bacterial regulation of gene expression in translation

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

When tryptophan levels within the cell are very low

trp repressor cannot bind to the operator site. Under these conditions, RNA polymerase transcribes the trp operon, and the cell expresses the genes required for the synthesis of tryptophan

When the tryptophan levels within the cell become high

tryptophan acts as a corepressor that binds to trp repressor. This causes a conformational change in trp repressor that allows it to bind to the trp operator site. This binding inhibits the ability of RNA polymerase to transcribe the operon. Therefore, when a high level of tryptophan is present within the cell—when the cell does not need to make more tryptophan—the trp operon is turned off.

small effector molecules

with regard to transcription, refers to a molecule that exerts its effects by binding to a regulatory transcription factor (a repressor or activator), causing a conformational change in the protein. regulatory proteins have one binding site where the protein binds to the DNA; the other is the binding site for the effector molecule.