GENETICS - CLUTCH CH.12 GENE REGULATION IN PROKARYOTES.

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1 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES!!

2 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES COCEPT: LAC OPERO 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 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 Page 2

3 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Page 3

4 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Page 4

5 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Lactose Glucose Lac Expression High Low Strongly Expressed Low High ot Expressed Low Low ot Expressed High High Somewhat Expressed Page 5

6 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Page 6

7 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Page 7

8 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 5. True or False: Glucose concentration can regulate the lac operon? a. True b. False Page 8

9 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES COCEPT: TRYPTOPHA OPERO AD ATTEUATIO 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 Attenuation Attenuation describes the process that uses tra trp 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 Page 9

10 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES P O Leader Sequence Operon Terminator Stops Transcription Anti-Terminator 2 3 Allows Transcription 1 4 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 tra trp 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 tra trp 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 Page 10

11 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES Low Tryptophan Ribosome Stalls Transcription continues P Ribosome O Operon UGGUGG High Tryptophan Ribosome Continues Transcription Stops P Ribosome O Operon UGGUGG UGA Stop Codon Page 11

12 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES TRAP Regulation of trp Operon Occasionally, other organisms have evolved ways of regulating the trp operon B. subtilis uses the Trp RA-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 Tryptophan Concentration: High P O Operon Transcription Stops TRAP w/ Tryptophan Tryptophan Concentration: Low 2 3 Transcription Continues P O 1 4 Operon TRAP/anti-TRAP Page 12

13 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 tras b. Tryptophan c. Lactose d. tra trp Page 13

14 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Page 14

15 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES COCEPT: LAMBDA BACTERIOPHAGE LIFE CYCLE REGULATIO 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 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 Page 15

16 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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,, ci, cro, and cii Lysogenic Cycle Genes Regulatory Genes Lytic Cycle Genes int xis ciii ci cro cii O P Q The first two mras transcribed are controlled via different - The 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 protein around, this will cause both the and cro genes to transcribe past the terminator - The protein is an anti-terminator, which allows transcription to take place Lysogenic Cycle Genes Regulatory Genes Lytic Cycle Genes int xis ciii ci cro cii O P Q T L P L P R T R1 Page 16

17 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES Decision to enter the lysogenic vs. lytic cycles The ci protein (lamda protein) controls entrance into the lysogenic cycle After the 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 Lysogenic Cycle Genes Regulatory Genes Lytic Cycle Genes int xis ciii ci cro cii O P Q cii cii T L P L P R T R1 P RE cii cii cii cii ci binds to two operator regions: OR and OL and inhibits them by preventing transcription of 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 Lysogenic Cycle Genes Regulatory Genes Lytic Cycle Genes int xis ciii ci cro cii O P Q Anti-cro ci ci P 1 T L P L P R T R1 ci X O L X O R P RE ci cii ci Anti-cro Anti-cro ci Anti-cro Page 17

18 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Lysogenic Cycle Genes Regulatory Genes Lytic Cycle Genes int xis ciii ci cro cii O P Q cro T L P L P R T R1 cro O L X P RE O R cro cro cro o ci = transcription 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 Lysogenic Cycle Genes Regulatory Genes Lytic Cycle Genes int xis ciii ci cro cii O P Q Anti-cro ci ci P 1 T L P L P R T R1 ci X O L X O R P RE ci cii ci Anti-cro Anti-cro ci Anti-cro Page 18

19 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 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 Page 19

20 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 3. Activation of which of the following genes leads to entrance into the lysogenic cycle? a., cro, and O genes b. O, P, and Q genes c. Int and xis genes d. ciii genes 4. The protein is an anti-terminator. What does this mean? a. The protein terminates transcription b. The protein allows for transcription to occur c. The protein terminates translation d. The protein allows for translation to occur Page 20

21 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES 5. Which of the following proteins is mainly responsible for entering the bacteriophage into the lysogenic cycle? a. protein b. Cro protein c. ci (Lambda) protein d. cii proteases Page 21

22 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES Page 22

23 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES COCEPT: RIBOSWITCHES Riboswitches are 5 UTR mra sequences that can bind small molecules and control gene expression of the mra 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 Page 23

24 GEETICS - CLUTCH CH.12 GEE REGULATIO I PROKARYOTES PRACTICE: 1. Riboswitches are made up of what type of molecule? a. RA b. DA c. Protein 2. Which of the following processes can riboswitches OT interfere with? a. Enhancers b. Transcription c. Splicing d. Translation Page 24