Exam 3

Question 1

What two criteria must the mechanisms of gene expression fulfill?

Answer 1

• They must be able to recognise environmental conditions in which they should activate or repress the transcription of the relevant genes
• They must be able to toggle on or off, like a switch, the transcription of each specific gene or group of genes

Question 2

What is the control of gene expression primarily governed by?

Answer 2

The control of gene expression is governed primarily by DNA-binding proteins that recognize specific control sequences of genes. (shown: the binding of the Lac repressor protein to the lac operator DNA)

Question 3

What controls transcription of a gene?

Answer 3

Regulatory proteins

Question 4

Binding sites for repressors in bacteria are called ________.

Answer 4

operators

Question 5

operator

Answer 5

In bacteria, operators are binding sites for repressor regulatory proteins

Question 6

promoter

Answer 6

The DNA sequence that participates in the DNA-protein interaction that determines where\ transcription begins

Question 7

activator

Answer 7

Activators are regulatory proteins that function by binding sequence-specifically to a DNA site located in or near a promoter and making protein–protein interactions with the general transcription machinery (RNA polymerase and general transcription factors), thereby facilitating the binding of the general transcription machinery to the promoter. The DNA site bound by the activator is referred to as an “activator site.” The part of the activator that makes protein–protein interactions with the general transcription machinery is referred to as an “activating region.” The part of the general transcription machinery that makes protein–protein interactions with the activator is referred to as an “activation target.”

Question 8

repressor

Answer 8

A DNA-binding repressor is a regulatory protein that blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking of expression is called repression.

Question 9

Define positive regulation of a gene

Answer 9

In positive regulation, an activator protein must bind to its target DNA site as a necessary prerequisite for transcription to begin

Question 10

Define negative regulation of a gene

Answer 10

In negative regulation, a repressor protein must be prevented from binding to its target site as a necessary prerequisite for transcription to begin

Question 11

genetic switch

Answer 11

Genetic switches control gene transcription. The on/off function of the switches depends on the interactions of several proteins with their binding sites on DNA. RNA polymerase interacts with the promoter to begin transcription. Activator or repressor proteins bind to sites in the vicinity of the promoter to control its accessibility to RNA polymerase.

Question 12

allosteric effector

Answer 12

Allosteric effectors control the ability of activator or repressor proteins to bind to their DNA target sites

Question 13

Describe an allosteric activator

Answer 13

Some activator or repressor proteins must bind to their allosteric effectors before they can bind DNA

Question 14

Describe an allosteric repressor

Answer 14

Some activator or repressor proteins bind DNA only in the absence of their allosteric effectors

Question 15

What must activators and repressors be capable of in order to function?

Answer 15

Both activator and repressor proteins must be able to recognize when environmental conditions are appropriate for their actions and act accordingly. Thus, for activator or repressor proteins to do their job, each must be able to exist in two states: one that can bind its DNA targets and another that cannot.

Question 16

X-Gal

Answer 16

X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. X-gal is an analog of lactose, and therefore may be hydrolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter then spontaneously dimerizes and is oxidized into 5,5’-dibromo-4,4’-dichloro-indigo, an intensely blue product which is insoluble. X-gal itself is colorless, so the presence of blue-colored product may therefore be used as a test for the presence of active β-galactosidase. This easy identification of an active enzyme allows the gene for β-galactosidase (the lacZ gene) to be used as a reporter gene in various applications.

Question 17

Blue-White screening

Answer 17

DNA inserts disrupt a gene (lacZ) in the plasmid that encodes an enzyme (β-galactosidase) necessary to cleave a compound added to the agar (X-gal) so that it produces a blue pigment. Thus, the colonies that contain the plasmids with the DNA insert will be white rather than blue (they cannot cleave X-gal because they do not produce β-galactosidase).

Question 18

lactose

Answer 18

Lactose is a disaccharide sugar derived from galactose and glucose

Question 19

DNA-binding domain (regulatory protein)

Answer 19

The site on a regulatory protein that binds DNA

Question 20

allosteric site (regulatory protein)

Answer 20

The allosteric site, acts as a sensor that sets the DNA-binding domain in one of two modes: functional or nonfunctional. The allosteric site interacts with small molecules called allosteric effectors

Answer 21

Question 22

Where does a prokaryotic activator bind?

Answer 22

The activator-binding site

Question 23

Where does a prokaryotic repressor bind?

Answer 23

At the operator

Question 24

positive regulation

Answer 24

An activator binds at the activator-binding site and transcription begins

Question 25

polycistronic

Answer 25

Describing a type of messenger RNA that can encode more than one polypeptide separately within the same RNA molecule. Bacterial mRNA is generally polycistronic.

Question 26

operon

Answer 26

a segment of DNA that encodes a multigenic mRNA as well as an adjacent common promoter and regulatory region

Question 27

What are the regulatory components of the lac operon?

Answer 27

- _The gene for the *lac* repressor, *lacI*_, that blocks the expression of genes *lacZ, lacY,* and *lacA*. Is **not part** of the *lac* operon, and proximity to the *lac* operon is not important for function. - _The *lac* promoter site, *lacP*_. Located in the *lac* operon, the site on the DNA to which RNA polymerase binds to initiate transcription of structural genes *Z, Y, A*. - _The *lac* operator site, *lacO*_. The *lac* repressor, *lacI*, binds to the operator site, located between *P* and *Z,* near the start transcription initiation

Question 28

What two enzymes does *Escherichia coli* require for the metabolism of lactose?

Answer 28

- β-galactosidase, encoded by *lacZ*, to cleave lactose molecule into glucose and galactose - β-galactoside permease, encoded by *lacA*, to transport lactose into the cell

Question 29

When is the *lac* operon transcribed?

Answer 29

Only in the presence of lactose

Question 30

What happens when lactose or an analog binds the protein product of *lacI*?

Answer 30

When lactose or its analogs bind to the repressor protein, the protein undergoes an allosteric transition, a change in shape. This slight alteration in shape in turn alters the DNA-binding site so that the repressor no longer has high affinity for the operator. Thus, in response to binding lactose, the repressor falls off the DNA: the *lac* operon is switched “on.”

Question 31

How does the presence of lactose enable the production of the structural genes of the *lac* operon?

Answer 31

When lactose or its analogs bind to the repressor protein (product of *lacI*), the protein undergoes an allosteric transition, a change in shape. This slight alteration in shape in turn alters the DNA-binding site so that the repressor no longer has high affinity for the operator. Thus, in response to binding lactose, the repressor falls off the DNA: the *lac* operon is switched “on.”

Question 32

The effect of lactose on the *lac* operon is an example of positive or negative control?

Answer 32

negative control (repression)

Question 33

induction

Answer 33

The releif of repression that allows expression. The behaviour of *lac* in the presence of lactose (an **inducer**) is an example.

Question 34

inducer

Answer 34

A compound that allosterically inactivates a repressor, allowing for the **induction** of gene transcription Lactose is an inducer of the *lac* operon

Question 35

IPTG

Answer 35

Isopropyl-β-D-thigalactoside An inducer of the *lac* operon that acts as a lactose analog for signalling molecules, but is unable to be metabolised by β-galactosidase and so remains at constant concentrations in the cell throughout the experiment.

Question 36

partial diploids

Answer 36

Bacteria that contain a second copy of some alleles on a secondary plasmid such as an F' factor

Question 37

constitutive mutation

Answer 37

A mutation that causes a gene to **continuously produces a protein** (and therefore may be produced in excess) due to a mutation in the regulatory gene, which is always expressed or impossible to turn off. Constitutive mutants are therefore those **strains that continuously produce a protein**, which in wild type is inducible. An example of constitutive mutant is a strain with a *lac* operon mutation that results in the transcription of the *lac* genes even if lactose is not present in the medium.

Question 38

cis-regulatory elements

Answer 38

cis-regulatory elements (CREs) are regions of non-coding DNA which regulate the transcription of nearby genes. The Latin prefix cis translates to “on this side”. CREs are found in the vicinity of the gene, or genes, they regulate. CREs typically regulate gene transcription by functioning as binding sites for transcription factors.

Question 39

*lacO*^C

Answer 39

A constitutive mutation in the *lac* operon in which the DNA sequence of the operator (*lacO*) region has been altered to show less affinity for the repressor (*lacI*), causing the transcription of the structural genes even in the absence of lactose. The superscript C denotes a cis-acting mutation, one in which the constitutive effects of *lacO*^C are restricted soley to those *lac* structural genes **on the same chromosome** as the *O*^C mutation.

Question 40

cis-acting

Answer 40

A segment of DNA that influences the transcription of genes **on the same chromosome** as it, as in the case of an operator, which acts as a protein binding site but makes no functional gene product

Question 41

trans-acting

Answer 41

A trans-acting gene product can regulate all structural *lac* operon genes, whether residing **on the same DNA molecule or on different ones** (in cis or in trans, respectively). A trans-acting product is a protein that is able to diffuse thoughout a cell and act on both operators of a partial diploid.

Question 42

cis-acting vs. trans-acting

Answer 42

- cis-acing genes are passive regions of DNA that allow proteins to bind and influence the expression of gene products of an adjacent transcription unit on the same chromosome (ex. *lacO* can only effect the transcription of the *lac* operon to which it is attatched) - trans-acting genes are actively transcribed regions of DNA that produce a diffusible protein product that is able to regulate transcription of any target transcription unit in the cell, even on different chromosomes (ex. *lacI* can repress **any** *lac* operon with a functional operator in a partial diploid).

Question 43

An operator is \_\_\_\_-acting, meaning it regulates the expression of \_\_\_\_\_\_\_\_\_\_\_, while a repressor is \_\_\_\_-acting, meaning it regulates the expression of \_\_\_\_\_\_\_\_\_\_.

Answer 43

An operator is _cis_-acting, meaning it regulates the expression of _an adjacent transcription unit_, while a repressor is _trans_-acting, meaning it regulates the expression of _any transcription unit containing the target DNA site in the cell_.

Question 44

super-repressor mutation

Answer 44

A mutation in the allosteric site of a repressor that prevents the binding of an inducer and thus perminently silences the target transcription unit under all regulatory (environmental) conditions.

Question 45

What is the name of the super-repressor mutation of the *lac* operon?

Answer 45

*lacI^S*

Question 46

What is the name of the constitutive mutation of the *lac* operon?

Answer 46

*lacO^C*

Question 47

What are the conserved features of the prokaryotic promoter sequence?

Answer 47

The -10 and -35 boxes

Question 48

cAMP-CAP complex binds *lac\_\_* in the presence of _____ \_\_\_\_\_.

Answer 48

cAMP-CAP complex binds *lac**_P_*** in the presence of _low glucose_.

Question 49

In the *lac* operon, where is the promoter region in comparison to the operator region? What binds at the promoter and operator? Where are these features in relation to the operon as whole? Where does transcription begin?

Answer 49

The promoter is upstream (towards 5') of the operator, both of which are immediately followed by *lacZ*, the first structural gene (encodes β-galactosidase) in the *lac* operon. The promoter is bound by the CAP-cAMP complex and RNA polymerase. The operator is bound by the protein product of *lacI*, the *lac* repressor. Transcription begins in the promoter region, the operator is transcribed at the 5' initiation end

Question 50

Where does transcription begin in the *lac* operon?

Answer 50

promoter region, *lacP*

Question 51

dyad symmetry

Answer 51

In genetics, dyad symmetry refers to two areas of a DNA strand whose base pair sequences are inverted repeats of each other. They are often described as palindromes. For example, the following shows dyad symmetry between sequences GAATAC and GTATTC which are reverse complements of each other.

Question 52

CAP

Answer 52

catabolite activator protein, encoded by the *crp* gene. The DNA-bound CAP is able to interact physically with RNA polymerase and increases that enzyme’s affinity for the lac promoter. By itself, CAP cannot bind to the CAP-binding site of the *lac* operon. However, by binding to cAMP, its allosteric effector, CAP is able to bind to the CAP-binding site and activate transcription by RNA polymerase.

Question 53

Where does RNA polymerase bind the *lac* operon? cAMP-CAP?

Answer 53

Question 54

What sites of the *lac* operon exhibit dyad symmetry?

Answer 54

*lac* operator (*lacO*) and the CAP-binding site inside the promoter (*lacP*) This rotational symmetry corresponds to symmetries within the DNA-binding proteins, many of which are composed of two or four identical subunits.

Question 55

Describe transcription by the *lac* operon under the presence of glucose and presence of lactose?

Answer 55

The presence of glucose means low cAMP, so low cAMP-CAP (positive control) = **repression of *lac* operon by catabolite repression** (glucose is a preferred catabolite, downstream from lactose catabolisation) The presence of lactose (inducer) means the repressor (*lacI,* a negative control) is unbound = **Induction of the *lac* operon by derepression**. Very low levels of *lac* mRNA

Question 56

Describe transcription by the *lac* operon under the absence of glucose and presence of lactose?

Answer 56

The absence of glucose means high cAMP, so high cAMP-CAP (positive control) bound = **Activation of *lac* operon by induction** (relief of catabolite repression) The presence of lactose (inducer) means the repressor (*lacI,* a negative control) is unbound = **Induction of the *lac* operon by derepression**. High levels of *lac* mRNA

Question 57

Describe transcription by the *lac* operon under the presence of glucose and absence of lactose?

Answer 57

The presence of glucose means low cAMP, so low cAMP-CAP (positive control) = **repression of lac operon by catabolite repression** (glucose is a preferred catabolite, downstream from lactose catabolisation) The absence of lactose (inducer) means the repressor (*lacI*) is bound (negative control) = **repression of *lac* operon** No *lac* mRNA

Question 58

initiator

Answer 58

The initiator element (Inr), sometimes also called initiator motif, is a core promoter that is similar in function to the Pribnow box (for prokaryotes) or the TATA box (for eukaryotes). It has the consensus sequence YYANWYY.[a][1] Similarly to the TATA box, the Inr element facilitates the binding of transcription Factor II D (TAF). TATA box and Inr are usually mutually exclusive.

Question 59

What does the binding of CAP do to DNA?

Answer 59

Bends it. DNA binds to multiple subunits of CAP, a reason for dyad symmetry in the DNA promoter sequence

Question 60

Features of the *ara* operon and what process does it regulate?

Answer 60

arabinose metabolisation In relation to the *lac* operon, * araC* is similar to *lacI* * araO* is similar to *lacO* * araI* where cAMP+CAP and *araC* bind * araP* is similar to *lacP* araB, araA, and *araD* are structural genes in the same manner as *lacZ, lacY,* and *lacA*

Question 61

The *ara* operon is an example of what form of gene regulation?

Answer 61

Dual positive and negative control In the presence of arabinose, both the CAP–cAMP complex and the *AraC*–arabinose complex must bind to *araI* in order for RNA polymerase to bind to the promoter and transcribe the *ara* operon (Figure 11-20a). In the absence of arabinose, the *AraC* protein assumes a different conformation and represses the ara operon by binding both to *araI* and to a second distant site, *araO*, thereby forming a loop (Figure 11-20b) that prevents transcription. Thus, the AraC protein has two conformations, one that acts as an activator and another that acts as a repressor. The on/off switch of the operon is “thrown” by arabinose. The two conformations, dependent on whether the allosteric effector arabinose has bound to the protein, differ in their abilities to bind a specific target site in the *araO* region of the operon.