Last Exam Part 13 Genetics

(What happens when an E. coli cell encounters the following conditions?) Glucose and lactose are present

. In the presence of glucose, cAMP levels are low. Thus, cAMP is not bound to CAP and CAP does not bind to the CAP site on the DNA. This is a negative signal that inhibits transcription. In the presence of lactose, allolactose binds to the lac repressor, releasing it from the operator. In this situation, the lac operon is off.

● (What happens when an E. coli cell encounters the following conditions?) Glucose is present, no lactose

. In this case, cAMP levels are low. At low cAMP levels, CAP does not bind to the CAP site on the DNA. This is a negative signal that inhibits transcription. Since there is no lactose, there is no allolactose, so the lac repressor is bound to the operator DNA sequence. In this situation, the lac operon is off.

Summary of lac Operon InductionWhat happens when an E. coli cell encounters lactose?

1. A small amount of lactose is transported into the cell by lactose permease. 2. Lactose is converted to allolactose by β-galactosidase. 3. The level of allolactose increases and allolactose binds to the lac repressor protein. 4. The lac repressor undergoes a conformational change and is released from the lac operator (lacO). 5. The lac operon is transcribed by the RNA polymerase core enzyme, producing abundant β-galactosidase, lactose permease, and galactoside transacetylase. 6. Lactose is metabolized rapidly into glucose and galactose to provide energy for the E. coli cell.

● (two factors involved in gene regulation in bacteria)Regulatory transcription factor protein.

The regulatory transcription factor that is responsive to glucose levels is called catabolite activator protein (CAP). The CAP protein binds to the CAP binding site in the DNA, which is located immediately upstream of the lac operon promoter (lacP).

(The lac operon contains the following DNA sequences and structural genes) lac terminator

The terminator is a DNA sequence involved in transcriptional termination. The lac operon is terminated by the rho (ρ)-dependent mechanism.

(The Jacob and Monod Experiment) When lactose is present, lactose is converted to

allolactose, binds to the two lac repressors, and releases the lac repressors from both copies of the operator. The lac operon on both the chromosome and on the F' plasmid is now expressed (tube 4). The expression of two copies of the lac operon produces two times the yellow color in tube 4.

The sequential use of sugars—first glucose, followed by lactose—is called

diauxic growth

(INDUCIBLE GENES)Repressor and activator proteins contain DNA binding domains and have additional domains that bind to small organic molecules called

effectors. When an effector binds to a repressor or an activator protein, the three-dimensional structure of the repressor or activator protein changes and this change in shape influences the activator and repressor protein's ability to bind to the DNA.

(The lac operon contains the following DNA sequences and structural genes) The lacI gene encodes the

lac repressor protein. The lac repressor binds to lacO and turns off the expression of the lac operon (in other words, the lac operon displays negative control via the lac repressor). The lacI gene is a constitutive or housekeeping gene and is therefore always transcribed.

(The lac operon contains the following DNA sequences and structural genes) lacA gene

lacA is the structural gene that encodes the enzyme galactoside transacetylase.

(The lac operon contains the following DNA sequences and structural genes) Near the lac operon is another gene, called _____that contains its own promoter and terminator.

lacI

(The lac operon contains the following DNA sequences and structural genes) Operator site (lacO)

lacO is the binding site for the lac repressor protein

(Mutant and Merozygote Strains of E. coli)Before we examine the experiment that determined which of the above hypotheses was correct, let us describe the strains of E. coli used by Jacob and Monod. These scientists used the lacI- strain described above, and they examined a very unusual strain of E. coli called a

merozygote, or partial diploid.

Typical operons contain a ___that serves as the binding site for σ factor, and the start site for transcription of the operon.

promoter

Inducible Genes Gene regulation in bacteria involves primarily controlling the initiation of transcription. This type of regulation involves the binding of

regulatory transcription factors to regulatory DNA sequences near the promoter region of a gene. These regulatory transcription factors function to either enhance or inhibit sigma (σ) factor and RNA polymerase core enzyme binding to the promoter.

The inducible proteins produced from regulated genes are

tightly controlled so that thousands of copies of the protein may be produced in certain environments, while only a few or no copies of the protein are produced in other environments. Regulated genes are advantageous to bacteria because they allow cells to respond to their environment, permitting the cell to survive adverse environmental conditions or compete for available resources, such as carbon or nitrogen. Other gene products produced by regulated genes are only needed during a limited stage of the life cycle, such as during cell division.

The merozygote strain used by Jacob and Monod contained lacI- on the chromosome and a wild-type copy of the gene (lacI+) on the F' plasmid; this E. coli strain was in essence a lacI+/lacI- heterozygote. The other DNA sequences within the lac operon (lacP, O, Z, Y, and A) were

wild- type and were found on both the chromosome and the F' plasmid (therefore, the E. coli cell was homozygous for these other DNA sequences).

● (Enzymes Involved in Lactose Metabolism in E. coli) Beta (β)-galactosidase.

β-galactosidase cleaves the disaccharide lactose, producing the monosaccharides galactose and glucose. β-galactosidase can also catalyze a side reaction that converts lactose into an isomer of lactose, called allolactose. Allolactose is an effector molecule. Specifically, allolactose is an inducer.

Regulatory transcription factor proteins include the following:

● Repressor proteins. Repressor proteins decrease how often transcription is initiated (exert negative control). ● Activator proteins. Activator proteins increase how often transcription is initiated (exert positive control).

Inducers can function in two different ways:

● The inducer can bind to a repressor. When the inducer binds to the repressor protein, the repressor is released from a binding site on the DNA, and transcription will occur. ● The inducer can bind to an activator. In this case, the activator protein cannot bind to the DNA unless the inducer is present. When the inducer binds to the activator protein, the activator can bind to the DNA and transcription will occur.

(Mutant and Merozygote Strains of E. coli)An example of a common type of plasmid is the

F plasmid that functions in DNA transfer between bacteria.

(The lac Operon in the Presence or Absence of Glucose)The lac operon can also be regulated by glucose. Glucose is the preferred carbon and energy source used by E. coli. The genes involved in glucose breakdown (catabolism) are expressed constitutively (always "on"). In the presence of glucose, the lac operon is not needed, so it is turned off in the presence of

glucose (catabolite repression). When glucose is limiting and lactose is present, this catabolite repression is alleviated, and the lac operon is turned on. Lactose is then used by the E. coli cell as a carbon and energy source.

(Mechanisms of Gene Regulation in Bacteria) Regulation of transcription

o Controlling how often transcription starts. Controlling how often transcription starts involves regulating how often σ factor (and the RNA polymerase core enzyme) binds to the promoter. Regulatory transcription factors (activator and repressor proteins) are involved in controlling how often transcription starts. The lac operon is an example of this type of gene regulation. o Attenuation. Attenuation involves activating transcription to begin producing a mRNA molecule; however, transcription is terminated prematurely before the entire mRNA is made.

In the presence of lactose, the few copies of β-galactosidase present produce allolactose. Allolactose is an inducer that binds to and changes the conformation of the lac repressor. When allolactose is present in a cell, the lac operon is induced as follows:

1. Allolactose binds to the lac repressor protein. The binding site for allolactose on the lac repressor is called the allosteric site. 2. The conformation of the lac repressor protein changes. 3. The lac repressor protein is released from lacO. 4. Sigma (σ) factor and the RNA polymerase core enzyme bind to lacP efficiently. 5. The structural genes of the lac operon (lacZ, lacY, and lacA) are transcribed.

What happens to an E. coli cell when lactose becomes limiting?

1. Allolactose levels become low due to the metabolism of allolactose. 2. Remaining allolactose is released from the lac repressor. 3. The lac repressor undergoes a conformational change and binds to the lac operator (lacO). 4. Sigma (σ) factor and the RNA polymerase core enzyme no longer bind efficiently to lacP. 5. The lac operon genes are turned off. 6. The excess β-galactosidase, lactose permease, and galactoside transacetylase enzymes are eventually degraded.

(The Jacob and Monod Experiment) The Jacob and Monod experiment compared a lacI- strain (lac operon is always expressed) to the lacI+/lacI- merozygote strain described above. The experiment was done as follows:

1. The mutant (lacI-) and the merozygote (lacI+/lacI-) strains were grown in separate flasks. 2. Each culture was split into two tubes, a control and an experimental tube. For example: ● Mutant strain (lacI-) oControl (tube 1) oExperimental (tube 2) ● Merozygote strain (lacI+/lacI-) oControl (tube 3) oExperimental (tube 4) 3. Lactose was added to the experimental tubes (tubes 2 and 4) to induce the genes involved in lactose breakdown. 4. The cultures were incubated to allow transcription and translation of the lactose metabolism genes. 5. The cells in each culture were then lysed using ultrasonic sound waves. 6. β-galactosidase levels in each of the four lysates were examined. β-galactosidase can convert the chemical β-O-nitrophenylgalactoside (β-ONPG), which is colorless, into galactose and O-nitrophenol (yellow). Note that if a yellow product is formed, β- galactosidase is present, meaning the lactose metabolism genes are expressed. 7. The O-nitrophenol levels in each test tube were measured using a spectrophotometer.

The lac Operon in the Absence or Presence of LactoseIn the absence of lactose, repression of the lac operon occurs as follows

1. lacI is a constitutive gene, meaning that it is always transcribed. Transcription of the lacI gene produces a lacI mRNA that is then translated to produce the lac repressor protein. 2. The lac repressor protein binds to the operator (lacO) DNA sequence. 3. Sigma (σ) factor and the RNA polymerase core enzyme do not bind efficiently to lacP when the lac repressor is bound to lacO. As a result, the three structural genes (lacZ, lacY, and lacA) of the lac operon are transcribed at a very low level, producing only a few (5-10) copies each of β-galactosidase, lactose permease, and galactoside transacetylase per cell.

(INDUCIBLE GENES)How do you turn genes from an off state to an on state? For example, how does a bacterium produce the enzymes necessary to metabolize the sugar lactose as a carbon source when lactose is present in the environment?

An effector molecule, called an inducer, causes transcription to increase

Enzymes Involved in Lactose Metabolism in E. coliLactose is a sugar that can be used as a carbon and energy source by the bacterium E. coli. Lactose breakdown by the bacterial cell involves three enzymes

Beta (β)-galactosidase, Lactose Permease, and Galactoside Transacetylase

The lac Operon in the Presence or Absence of Glucose) When the CAP protein binds to cAMP, the CAP protein can then bind to the

CAP site in the DNA and lac operon transcription is activated. In fact, for sigma (σ) factor and the RNA polymerase core enzyme to bind efficiently to the lac promoter and therefore transcribe the lac operon, CAP must be bound to the CAP site.

Lactose metabolism requires regulating genes within the lactose (lac) operon. The lac operon contains the following DNA sequences and structural genes

CAP site, lac promoter, operator site(lacO), lacZ gene, lacY gene, lacA gene, and lac terminator.

The merozygote strain that Jacob and Monod used in their experiments contained a modified F plasmid called an

F' plasmid, which contained a lacI gene and the lac operon genes. Thus, E. coli cells that harbor an F' plasmid are merozygotes (partial diploids), containing a copy of lacI and the lac operon on the chromosome and a second copy of lacI and the lac operon on the F' plasmid.

(Enzymes Involved in Lactose Metabolism in E. coli) Galactoside transacetylase.

Galactoside transacetylase is thought to convert atypical isomers of lactose into forms that can be metabolized by β-galactosidase.

(The Jacob and Monod Experiment) Merozygote strain.

In the merozygote strain, no yellow color was produced in the absence of lactose; however, two times the yellow color was produced in the presence of lactose. The lac operon is not transcribed when lactose is absent (tube 3) because the lacI+ gene on the F' plasmid produces lac repressor proteins that bind to both copies of lacO, and thus, inhibit expression of both the chromosomal lac operon and the F' plasmid lac operon. Remember that bacteria do not have a nuclear membrane, so both the host chromosome and the F' plasmid are found in the cytoplasm. This lac repressor protein diffuses throughout the cytoplasm of the cell and can bind to any lac operator. Because the lac repressor can bind to any operator in the cell, the lac repressor is said to be an example of a trans-acting factor.

(What happens when an E. coli cell encounters the following conditions?) Glucose is absent, lactose is present

In this case, cAMP levels are high. cAMP binding to CAP changes the conformation of CAP, allowing CAP to bind to the CAP site on the DNA. This serves as a positive signal for transcription. When lactose is present, allolactose is present. The lac repressor binds to allolactose and therefore is released from the operator DNA site. In this situation, the lac operon is "on".

(What happens when an E. coli cell encounters the following conditions?) No glucose or lactose.

In this environment, cAMP levels are high, and the cAMP is bound to CAP. CAP is targeted to the CAP site on the DNA. This is a positive signal for transcription. However, in the absence of lactose, there is no allolactose, so the lac repressor is bound to the operator DNA sequence, preventing transcription. In this situation, the lac operon is off.

● (two factors involved in gene regulation in bacteria)Effector molecule.

It would be reasonable to assume that glucose is the effector molecule involved in catabolite repression; instead, the effector involved is cyclic AMP (cAMP)

(Constitutive Expression of the lac Operon )How could this phenotype be explained?

Jacob and Monod suggested that the lacI- phenotype could be explained in two ways: ● The lacI- mutation produces a defective protein that activates transcription under all environmental conditions (constitutive activator hypothesis). ● The lacI- mutation produces a defective protein that fails to inhibit transcription (defective repressor hypothesis).

● (Enzymes Involved in Lactose Metabolism in E. coli )Lactose permease.

Lactose permease is a membrane protein involved in the transport of lactose from the environment into the cytoplasm of the E. coli cell.

(The lac operon contains the following DNA sequences and structural genes) CAP site.

The CAP site is a DNA sequence that serves as the binding site for an activator protein called the catabolite activator protein (CAP).

(The Jacob and Monod Experiment) Experimental Results Show that lacI+ Produces a Diffusible Repressor Protein Mutant strain (lacI-).

Yellow color was observed in both test tube 1 and in tube 2. Thus, in the lacI- strain, the lactose metabolism genes are expressed in the absence and in the presence of lactose.

(The lac Operon in the Presence or Absence of Glucose) cAMP is produced from ATP by an enzyme called

adenylyl cyclase. When glucose is present, adenylyl cyclase activity is inhibited, and cAMP levels decrease. When glucose levels are low, adenylyl cyclase activity increases, producing higher levels of cAMP.

Operons contain the following:

an operator DNA sequence where a repressor protein binds, an activator binding site DNA sequence where an activator protein binds, structural genes that encode proteins, and a terminator that signals the end of transcription. Recall that the terminators in bacteria work either using the rho (ρ)-dependent or ρ-independent mechanism.

François Jacob and Jacques Monod identified a mutant strain of E. coli that they named lacI-. In this lacI- mutant strain, the enzymes involved in lactose metabolism are always produced, even in the absence of lactose. Thus, the lacI- mutation produces constitutive expression of the lac operon and is a

constitutive mutation

Some bacterial genes are always transcribed. These genes that are always on are called

constitutive or housekeeping genes.

Note that constitutive genes produce

constitutive proteins. The products of these genes are required for the normal functioning of the bacterial cell, the so-called housekeeping functions.

Operons allow proteins that are involved in certain biochemical pathways (for example, lactose metabolism, tryptophan biosynthesis) to be

controlled in a coordinated way. When an operon is transcribed, a long polycistronic mRNA is produced that contains the coding regions for multiple types of proteins.

Regulated genes produce

inducible proteins.

● (The lac operon contains the following DNA sequences and structural genes) lac promoter (lacP)

lacP contains the -35 sequence, the -10 sequence, and the +1 site. lacP determines where transcription of the lac operon will begin, serving as the binding site for σ factor. Recall that σ factor targets the RNA polymerase core enzyme to the +1 site.

(The lac operon contains the following DNA sequences and structural genes) lacY gene.

lacY is the structural gene that encodes the enzyme lactose permease

(The lac operon contains the following DNA sequences and structural genes) lacZ gene.

lacZ is the structural gene that encodes the enzyme β-galactosidase.

The Lactose (lac) OperonFrançois Jacob and Jacques Monod first described transcriptional regulation in bacteria by studying

lactose metabolism in E. coli. Jacob and Monod won the Nobel Prize for their work.

●( Mechanisms of Gene Regulation in Bacteria) Posttranslational regulation

o Feedback inhibition. Feedback inhibition is a situation in which the product of a metabolic pathway inhibits the first enzyme in the pathway.o Covalent modification. Covalent modification involves altering the structure and function of a protein by attaching phosphate groups, methyl groups, sugars, or lipids.

(Mechanisms of Gene Regulation in Bacteria) Regulation of translation.

o Translation repressor proteins. Translation repressor proteins prevent the initiation of translation. These repressor proteins bind to the Shine-Dalgarno sequence, preventing the 16S rRNA component of the ribosome from binding to the mRNA. o Antisense RNA. Antisense RNA molecules are produced by the E. coli cell to bind to the mRNA, forming a double-stranded RNA (dsRNA) molecule. Antisense RNA molecules form hydrogen bonds with the Shine-Dalgarno sequence, preventing the 16S rRNA component of the ribosome from binding to the mRNA. Antisense RNA molecules are examples of noncoding RNAs (ncRNAs). ncRNAs are not translated to make protein products.

In bacteria, a group of structural genes (genes that produce proteins) can be under the control of a single group of regulatory DNA sequences, a single promoter, and a single terminator. This grouping of structural genes is called an

operon

(Mutant and Merozygote Strains of E. coli)Bacteria typically have a single circular chromosome. However, bacteria often contain small circular DNA molecules in addition to the chromosome. These small DNA molecules are called

plasmids

Gene expression

refers to processes that activate a gene, producing first, a mRNA molecule, and eventually, a functional protein product.

The expression of some bacterial genes changes under different environmental conditions; these genes are called

regulated genes.

How is the lac operon repressed (turned off) by glucose? Recall that there are (two factors involved in gene regulation in bacteria):

regulatory transcription factor and effector molecule

lac operon in E coli regulates

the genes that control lactose metabolism in a bacterial cell.

(Repressible Genes )How do you turn genes from an on state to an off state? For example, how does a bacterium stop producing the enzymes required for the biosynthesis of the amino acid, tryptophan, when there is plenty of tryptophan in the cell? The presence of an effector molecule may inhibit transcription in two ways

● An effector molecule called a corepressor binds to a repressor protein. Without the corepressor, the repressor protein does not bind to the DNA. When the corepressor binds the repressor, a conformational change occurs in the repressor. The repressor can then bind to the DNA and prevent transcription. ● An effector molecule, called an inhibitor, binds to an activator. In this case, the activator protein is normally bound to the DNA and activates transcription. When the inhibitor binds to the activator, a conformational change causes the activator to be released from the DNA, and transcription ceases.