GENETICS - CLUTCH CH.12 GENE REGULATION IN PROKARYOTESlightcat-files.s3.amazonaws.com/packets/admin_genetics-18-genetic… · a. Translation stalls, forming anti-termination sequence

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GENETICS - CLUTCH

CH.12 GENE REGULATION IN PROKARYOTES

CONCEPT: LAC OPERON

● An operon is a group of genes with similar functions that are transcribed together □ There are many __________________________ of an operon (PROG)

- Promoter: Region where transcription initiator binds to initiate transcription

- Repressor: A protein that can repress transcription of the operon

- Operator: Region where the repressor binds. The “on/off” switch

- Genes: The genes that are transcribed together

EXAMPLE:

● An lac operon was the first operon discovered. It was discovered by Jacob, and Monod in the 1960s □ The lac operon encodes ___________________ that breakdown and process lactose

- LacZ: Encodes beta-galactosidase which converts lactose into glucose and galactose

- LacY: Ecodes permease, which allows lactose to enter into the cell

- LacA: Encodes transacetylase, which has an unknown function but is crucial for lactose processing

EXAMPLE:

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Lac operon regulation

● The lac operon responds differently to different lactose _________________________ □ When the lactose concentration is high:

- Lactose binds to the repressor, causing it to be removed from the operator

- When the repressor is removed, transcription of the operon can take place

- The lac operon genes are made, and then can act to breakdown lactose

□ When the lactose concentration is low:

- Lactose doesn’t bind to the repressor, causing it to remain on the operator

- Transcription does not take place

EXAMPLE:

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● The lac operon can also respond to glucose ________________________ □ The catabolite activator protein (CAP) represses the lac operon when glucose is present

- When glucose is present it inhibits activity of adenyl cyclase, which works to create cAMP

- When the cAMP concentration is high it binds CAP, and when it is low it doesn’t bind to CAP

- CAP/cAMP complex binds CAP site upstream of the lac promoter and activates transcription

- When glucose is high, it will inhibit cAMP production, and will not activate transcription

- When glucose is low, there will be high cAMP production, which will activate transcription

EXAMPLE:

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Summary of lac operon expression ● Therefore the lac operon has different functions depending on the ____________________ of glucose and lactose □ If glucose and lactose are both present, the cell would prefer to utilize glucose, because it is simpler

- But, if you only have lactose, then the cell has to break down lactose to generate glucose

EXAMPLE:

Lactose Glucose Lac Expression High Low Strongly Expressed Low High Not Expressed Low Low Not Expressed High High Somewhat Expressed

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PRACTICE:

1. Which of the following is not a part of an operon? a. Promoter b. Repressor c. Operator d. Enhancer

2. The lac operon encodes genes that are responsible for what? a. Synthesizing more lactose b. Breaking down lactose c. Carrying lactose to the mitochondria d. Converting lactose into cellulose

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3. What happens to the lac operon when lactose concentration is high? a. The lac operon is activated, and lactose is synthesized b. The lac operon is activated, and lactose is broken down c. The lac operon is repressed, and lactose is synthesized d. The lac operon is repressed, and lactose is broken down

4. What happens to the lac operon when the CAP/cAMP complex binds to the CAP binding site? a. The lac operon genes are transcribed b. The lac operon genes are suppressed c. Lactose is synthesized

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5. True or False: Glucose concentration can regulate the lac operon? a. True b. False

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CONCEPT: TRYPTOPHAN OPERON AND ATTENUATION

● The Trp operon encodes genes that synthesize and process the amino acid tryptophan □ The trp operon is regulated by tryptophan in _______________- ways: a repressor and attenuation

- Cytoplasmic tryptophan acts as a corepressor when regulating the trp operon

- Tryptophan binds to a repressor, which then binds to the operator and represses transcription

EXAMPLE:

Attenuation

● Attenuation describes the process that uses tRNAtrp levels to ______________________ the trp operon

□ When tryptophan levels are high, attenuation turns off the trp operon

□ The trp operon has a leader sequence of 100+ nucleotides prior to the start site but after the promoter

- The leader sequence can form different types of secondary structures by combining 4 small sequences

- These sequences are called regions 1, 2, 3, and 4

□ A terminator structure forms when 1 and 2 form a loop and 3 and 4 form a loop

- Terminates transcription

□ An anti-terminator structure forms when 2 and 3 form a loop

- Allows transcription to continue

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EXAMPLE:

□ The leader sequence can also be used to control translation because it contains many tryptophan ___________

- If tryptophan levels are low, there will be very little tRNAtrp and therefore translation stalls

- When translation stalls, 2 and 3 forms a loop forming an anti-termination sequence

- Anti-termination sequence promotes transcription

- If tryptophan levels are high, there will be enough tRNAtrp and translation continues

- Translation continues until it reaches a stop codon at the end of region 1

- Then region 3 forms a loop with 4 and acts as a termination sequence – stopping transcription

P O Leader Sequence Operon

1 2 3 4

1 2 3 4

1

2 3

4

Terminator

Stops Transcription

Anti-Terminator

Allows Transcription

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EXAMPLE:

P O

UGGUGG

Ribosome

Low TryptophanRibosome Stalls

1

2 34 Operon

Transcription continues

P O

UGGUGG

Ribosome

High TryptophanRibosome Continues

1 2

3 4Operon

Transcription Stops

UGA

Stop Codon

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TRAP Regulation of trp Operon

● Occasionally, other organisms have evolved _____________________ ways of regulating the trp operon □ B. subtilis uses the Trp RNA-Binding Attenuation Protein (TRAP)

- TRAP binds to multiple tryptophan molecules

- When tryptophan concentration is high TRAP is saturated, and binds to the leader sequence

- This forms the terminator configuration and prevents transcription

- A second protein anti-TRAP binds to TRAP when tryptophan is low

- This allows fort he formation of the anti-terminator configuration and promotes transcription

- The TRAP/anti-TRAP regulatory method is sensitivite to a wide variety of tryptophan concentrations

EXAMPLE:

Tryptophan Concentration: High

Tryptophan Concentration: Low

P O Operon1 2 3 4

TRAP w/ Tryptophan

P O Operon1

2 3

4

TRAP/anti-TRAP

Transcription Stops

Transcription Continues

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PRACTICE:

1. Tryptophan regulates the trp operon by doing what? a. Activating the operon and synthesizing more tryptophan b. Activating the operon and breaking down tryptophan c. Repressing the operon and inhibiting further tryptophan synthesis d. Repressing the operon and inhibiting breakdown of tryptophan

2. Attenuation uses what molecule to regulate the trp operon? a. All tRNAs b. Tryptophan c. Lactose d. tRNAtrp

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3. What is the name of the sequence responsible for regulating the trp operon through attenuation? a. Leader sequence b. Regulator sequence c. Terminator sequence d. Anti-terminator sequence

4. If tryptophan levels are low, attenuation does what to the trp operon? a. Translation stalls, forming anti-termination sequence which promotes transcription b. Translation is activated and promotes tryptophan creation c. Transcription is inhibited d. A termination structure is formed blocking translation

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CONCEPT: LAMBDA BACTERIOPHAGE LIFE CYCLE REGULATION

● Bacteriophages are viruses that infect bacteria □ Bacteriophages have _______________________ life cycles

- Lytic cycle is a period of active virus replication, which bursts the host cell

- Lysogenic cycle is a period where the virus integrates into the genome, and is silent

□ The bacteriophage chromosome contains two sets of genes: One for each the lytic and lysogenic cycle

- Regulating the expression of these genes determines which cycle the bacteriophage enters

EXAMPLE:

□ If there are good growth conditions, there will be more cro protein which leads to lytic cycle

□ If there are poor growth conditions, there will be more lambda protein (cI) which leads to the lysogenic cycle

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Mechanism of Regulation

● The regulatory genes are physically __________________________ on the bacterial chromosome □ The lytic cycle involves the O, P, and Q genes

□ The lysogenic cycle includes the int and xis genes

□ In between the lytic and lysogenic cycle genes there four genes: cIII, N, cI, cro, and cII

EXAMPLE:

□ The first two mRNAs transcribed are controlled via different _______________________

- The N gene is transcribed by the PL promoter. It is terminated by TL

- The cro gene is transcribed by the PR promoter. It is terminated by TR1

- These genes are transcribed in reverse of the other

□ If there is a lot of N protein around, this will cause both the N and cro genes to transcribe past the terminator

- The N protein is an anti-terminator, which allows transcription to take place

EXAMPLE:

int xis cIII N cI cro cII O P Q

Lysogenic Cycle Genes

Lytic Cycle GenesRegulatory Genes




PLTL PR TR1

NN

NN

NNN

NN

NN

N

NN

N

N

N

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Decision to enter the lysogenic vs. lytic cycles

● The cI protein (lamda protein) controls entrance into the lysogenic cycle □ After the N gene blocks termination, the cII protein is ____________________

- The cII protein activates the PRE promoter that sits at the TR1, and transcribes anti-cro and cI

- Anti-cro is the reverse of the cro gene

- cI (lambda protein) controls entering the lysogenic cycle

EXAMPLE:

□ cI binds to two operator regions: OR and OL and inhibits them by preventing transcription of N and cro and longer

- cI activates the PRM promoter which promotes transcription of more cI protein

- cI activates the P1 promoter which activates the transcription of int and xis

EXAMPLE:




PLTL PR TR1N N

cIIcII

cIIcII cII

cII

PRE

OROL




PLTL PR TR1N N

cIIPRE

cI

Anti-cro

cI

Anti-cro

cIAnti-cro

cI

Anti-cro

P1

cI cI

X X

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● The cro protein controls ______________________ into the lytic cycle □ When there is more cro around it binds to OL and OR and repress them

- When OL and OR are inhibited, this inhibits PL and PRM

- This lowers the amount of cI in the cell – which means it wont inhibit anything

- A lot of lytic cycle genes are created

EXAMPLE:

● In bacterial cells, proteases which destroy the cII protein, control _________________ into the lytic or lysogenic cycle □ In good growth conditions there are plenty of cII proteases that degrade cII

- Less cII means that the P1 promoter wont be activated by cI, and therefore promote the lytic cycle

□ In poor growth conditions there are not many cII proteases, meaning that there are high levels of cII

- High levels of cII will activate cI and P1 and activate the lysogenic cycle

EXAMPLE:




PLTL PR TR1

N N

cro

crocro

OROL crocro

PREX

No cI = transcription

OROL




PLTL PR TR1N N

cIIPRE

cI

Anti-cro

cI

Anti-cro

cIAnti-cro

cI

Anti-cro

P1

cI cI

X X

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PRACTICE: 1. In which of the following life cycles does a bacteriophage integrate itself into the host genome?

a. Lysogenic cycle b. Integrative cycle c. Lytic cycle d. Subdued cycle

2. In good growth conditions the bacteriophage is more likely to enter into which life cycle? a. Lysogenic cycle b. Integrative cycle c. Lytic cycle d. Subdued cycle

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3. Activation of which of the following genes leads to entrance into the lysogenic cycle? a. N, cro, and O genes b. O, P, and Q genes c. Int and xis genes d. cIII genes

4. The N protein is an anti-terminator. What does this mean? a. The N protein terminates transcription b. The N protein allows for transcription to occur c. The N protein terminates translation d. The N protein allows for translation to occur

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5. Which of the following proteins is mainly responsible for entering the bacteriophage into the lysogenic cycle? a. N protein b. Cro protein c. cI (Lambda) protein d. cII proteases

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CONCEPT: ARABINOSE OPERON

●The arabinose operon encodes genes that control the breakdown of arabinose ___________________ □ The araI (initiator) and araO regions are responsible for regulating the ara operon

- When arabinose is present: araC protein binds to araI and initiates transcription

- cAMP/CAP complex also binds this region to help initiate transcription

- When arabinose is not present: araC binds to araI and araO and represses transcription

- This confirmation forms a DNA loop and prevents the polymerase from binding

EXAMPLE:

PRACTICE: The genes in the arabinose operon are responsible for what? a. Synthesizing arabinose b. Breaking down arabinose c. Converting arabinose into starch d. Synthesizing sugar

PRACTICE: When arabinose is present, the arabinose operon is what? a. Active – meaning the genes are being transcribed b. Inactive – meaning the genes are not being transcribed c. Nothing, arabinose does not control the arabinose operon

araIaraO CAP

Operon

araCarabinose

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CONCEPT: RIBOSWITCHES

● Riboswitches are 5’ UTR mRNA sequences that can bind small molecules and control gene expression of the mRNA □ Riboswitches control the expression of ______________________________

□ There are two main domains in a riboswitch

- An aptamer is the sequence that binds the small molecule ligand

- An expression platform forms secondary structures which regulate gene expression

- A terminator structure is the structure formed by binding the small ligand

□ Riboswitches can interfere with transcription, splicing, and translation

- Transcription termination is controlled by a terminator structure

- Translation termination is controlled by a terminator structure blocking the ribosome binding site

EXAMPLE:

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PRACTICE:

1. Riboswitches are made up of what type of molecule? a. RNA b. DNA c. Protein

2. Which of the following processes can riboswitches NOT interfere with? a. Enhancers b. Transcription c. Splicing d. Translation

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GENETICS - CLUTCH CH.12 GENE REGULATION IN PROKARYOTESlightcat-files.s3.amazonaws.com/packets/admin_genetics-18-genetic… · a. Translation stalls, forming anti-termination sequence