Prokaryotic Transcriptional Regulation (Theme 3: Module 2)

Question 1

Metabolic shift (in E.Coli)

Answer 1

occurs from switching from glucose to lactose as a fuel source

-tightly regulated in bacteria such that they are able to quickly upregulate the expression of genes that produce lactose-metabolizing enzymes when glucose is no longer available, and lactose is predominant nutrient source

Question 2

At what level is the control of enzyme production regulated:

Answer 2

transcriptional level

-transcription of many genes in the E.Coli genome are turned on to accommodate for this change in nutrient source

Question 3

How is metabolic shift enabled?

Answer 3

environmental cues

As E.Coli cells utilize the nutrients that are available in the environment, the bacterial cells are able to detect environmental cues that facilitate this transition from glucose to lactose metabolism

Question 4

As E.Coli cells transition from glucose to lactose metabolism, there is a concomitant increase in the amount of:

Answer 4

-detectable beta-galactosidase
-lactose permease proteins

Not detectable when: E.Coli Cells are utilizing glucose as a primary nutrient source

Some are expressed: as they are required for the transport and metabolism of lactose

Genes are not expressed until glucose is fully depleted from the growth medium

Question 5

Once glucose is depleted, and bacterial cells are starting to utilize lactose:

Answer 5

there is an increase in the expression of the beta-galactosidase and lactose permease proteins (glucose inhibits the expression of these gene products, lactose induces their expression once glucose is depleted)

Question 6

Environment where the expression of beta-galactosidase and lactose occurs in response to lactose metabolism:

Answer 6

medium where glucose nutrients have been depleted

-proteins are produced in response to environmental cues and allow for effective digestion of lactose

Question 7

Lactose Permease

Answer 7

transport protein that sits in the bacterial cell membrane and allows for the transport of lactose into the bacterial cells

Question 8

Beta-Galactosidase Protein

Answer 8

cytoplasmically situated bacterial enzyme that cleaves the imported lactose into glucose and galactose

Question 9

key advantage of the organization of the prokaryotic genome:

vs eukaryotes

Answer 9

groups of related genes w/similar functions can often be found clustered together into operons

Diff from eukaryotes: each gene has its own promoter and enhancers. This leads to the ability to control the transcription of the whole gene cluster as one unit

Question 10

Francois Jacob and Jacques Monod

Answer 10

discovered the operon model - the basic model for gene expression in bacteria

-in bacteria, groups of functionally related genes are organised into transcriptional units along the bacterial chromosome
-these gene clusters are controlled by a single “on-off” switch that can control the transcription of the clustered genes
-this coordination is mediated by an operon

Question 11

Bacterial Operon consists of:

Answer 11

-a promoter
-an operon (on/off switch)
-the coordinated gene cluster whose products will function in a common pathway or cellular response

Question 12

Operator

Answer 12

a sequence of nucleotides near the start of the operon that can be regulated to allow or inhibit transcription

-when the operon is not bound to any transcriptional inhibitor, then the RNA polymerase can attach to the promoter and transcribe the genes in the operon

Question 13

Transcription in bacteria can give rise to one long

Answer 13

mRNA molecule (polycistronic mRNA) that can code for many proteins

Question 14

During translation bacterial cells can produce…

Answer 14

separate polypeptides

because the polycistronic mRNA is punctuated w/start and stop codons that signal where the coding sequence for each polypeptide begins and ends

Question 15

Lac Operon controls:

Answer 15

regulation of beta-galactosidase and lactose permease expression (in E.Coli cells)

Question 16

Regulatory sequences of transcription that exist within the Lac Operon:

Answer 16

-the promoter: binds the RNA polymerase complex

-the operator (lac O): a binding site for a repressor protein that is expressed by lacl coding sequence

Question 17

Two main structural genes that code for the primary proteins that are needed to facilitate lactose metabolism:

Answer 17

1. lacY gene: codes for lactose permease transport protein which will embed itself in the cell membrane and allow for the import of lactose into the bacterial cell
2. lac Z gene: codes for B-galactosidase that will be able to cleave the disaccharide lactose into glucose and galactose

Question 18

Lac I gene:

Answer 18

controls the expression of the lacZ and lacY genes.

-this gene codes for a repressor protein which can bind to the operator and inhibit transcription from occurring

Question 19

Negative Transcriptional Regulation

Answer 19

the ability of a repressor protein to halt transcription

-during it: a repressor protein will bind to the operator region of the operon, this will result in turning off transcription since the RNA polymerase is no longer able to bind to the promoter region of the operon

Question 20

How is the lactose operon regulated?

Answer 20

Negatively regulated ; which means that the transcription of genes that are required for lactose metabolism is turned off

turns off how?
the repressor protein, encoded by the lacl gene is constitutively expressed at low levels.

-because of this, the repressor protein is able to bind w/the lacO operator region and as a result, the RNA polymerase complex is not able to bind to the promoter

-this is the state when E.Coli cells are exposed to glucose, the preferred nutrient source, and in this state there is no B-galactosidase or lactose permease produced

Question 21

Repressor protein (creates DNA loop) :

Answer 21

a tetrameric protein which is made of 4 identical protein subunits that binds tightly to the operator regions on the lac operon DNA. Once all 4 subunits bind to the lac operon DNA, the DNA is twisted into a loop

Question 22

what happens when DNA loop is created by the repressor protein?

Answer 22

RNA polymerase is not able to bind to the lac operon promoter, as a result, transcription is inhibited or repressed

ex: when E.Coli cells are grown in an environment w/glucose being used as the primary nutrient source, presence of glucose is the bacterial cell growth medium will facilitate the constitutive expression of the repressor protein and this inhibit the expression of proteins that are needed for lactose metabolism

Question 23

How does lactose inhibit the repressor protein?

Answer 23

Lactose acts as an inducer molecule: by binding to the repressor proteins on the lac operator and causing a change in the conformation of the repressor

Question 24

To inhibit repressor function, lactose is able to…

Answer 24

bind to specific binding sites on the repressor protein

-leads to a conformational change in the repressor which no longer allows it to attach to the operator sequences in the lac operon DNA

-when the operator is not bound by the transcriptional inhibitor, then the RNA polymerase can attach to the promoter and transcribe the genes in the operon to produce both the beta-galactosidase and the lactose permease that are required for the metabolism of lactose

Question 25

Positive Regulation of the Lac Operon

Answer 25

occurs in the absence of glucose, promotes the production of beta-galactosidase and lactose permease

-decrease in environmental glucose levels results in an increase in intracellular cAMP inside the bacterial cells - which contributes to positive regulation of lac operon

Question 26

Concentration of cAMP in bacterial cell is a good indicator of:

a)when glucose levels are high..
b)when glucose levels are low..

Answer 26

the general nutritional state of the E.Coli cells

a)there is an inhibition of the enzyme adenylyl cyclase, which catalyses the production of cAMP from ATP (results in low levels of intracellular cAMP)

b)bacterial cells will accumulate high levels of cAMP due to increased activity of the adenylyl cyclase enzyme

Question 27

Positive regulation of Lac Operon involves a protein called:

Answer 27

CRP or CAP (cAMP receptor protein) , which when bound by cAMP will bind to a different site on the bacterial DNA called the CRP-cAMP or CAP-cAMP binding site

Question 28

amount of intracellular camp determines:

Answer 28

whether a transcriptional activator will bind to this site and therefore the degree of positive regulation of the lac operon

Question 29

Negatively regulated transcription

Answer 29

a repressor protein will bind to the operator region of the operon, this will result in turning off transcription since RNA polymerase is no longer able to bind to the promoter region of the operon, however this can be inhibited by inducer proteins

Question 30

Inducer Proteins

Answer 30

are able to bind to the repressor proteins and prevent their binding to the DNA

inducer proteins become inducers of genes

Question 31

Positively regulated transcription

Answer 31

a transcriptional activator protein binds to an activator site on the DNA

activator site can be: upstream, downstream, overlap w/the promoter

when the activator binds to the DNA, it allows for the recruitment of RNA polymerase to the promoter and then the initiation of transcription
(transcription could not occur if activator is not present or is not able to bind to the activator binding site)

Question 32

Levels of Lac Operon mRNA:

Answer 32

Highest level: point C(end of graph) because the bacteria are actively utilizing lactose as a nutrient source, as a result, are transcribing the lac operon gene products to be able to produce lactose permease and B-galactosidase

Lowest levels: Point B (middle)
because it is the transition time point where glucose has been used up and lac operon transcription is initiating

No lac operon mRNA: Point A (bottom/beginning of graph)
because the bacteria are still actively utilizing glucose as a nutrient source in this environment that contains both glucose and lactose. since glucose is the preferred nutrient source, lac operon transcription is repressed until a time when there is no longer any glucose available and the metabolic shift towards lactose metabolism is required

Question 33

High extracellular glucose concentration:

Answer 33

an inhibition on the adenylyl cyclase enzyme, results in low levels of cAMP

-even in the presence of lactose, the CRP-cAMP complex will not bind the lac operon, there will be lower levels of transcription

In this manner, E.coli cells are able to utilize all glucose in the environment when both glucose and lactose are present, after which lactose will only be utilized once glucose is fully depleted

Question 34

cAMP signals the nutritional state of E.Coli cells:

Answer 34

as bacteria transition from glucose to lactose metabolism the low levels of glucose signal an increase in cAMP to activate the positive regulator (cRP-cAMP) and the presence of lactose binds the receptor protein LaCl to allow transcription of the operon to occur