Lecture 15

Question 1

What are operons?

Answer 1

multigene transcriptional units occurring mostly in prokaryotes… functional unit of genomic DNA that contains a cluster of genes with related functions

Question 2

Why are eukaryotes rarely organized into operons?

Answer 2

their complex cellular structure, particularly the presence of a nuclear membrane which separates transcription and translation

Question 3

What is bacterial induction?

Answer 3

the synthesis of enzymes in response to the appearance of a specific substrate… cell produces necessary enzyme only when its corresponding substrate is present and this is a form of cellular regulation that conserves energy by not producing enzymes that are not needed

Question 4

What is the Lac operon?

Answer 4

cluster of genes in bacteria that allows them to metabolize lactose (a sugar) for energy

Question 5

What happens to the lac operon in the absense of lactose?

Answer 5

lac operon is turned off because the lac repressor protein binds to the operator region which blocks RNA polymerase from transcribing genes for lactose metabolism

Question 6

What does CAP do?

Answer 6

CAP is a regulatory protein that enhances transcription when glucose is low and its activity is dependent on cAMP
it recruits RNA polymerase and helps core RNA polymerase bind tightly to the promoter in association to the cAMP protein

Question 7

What happens to the lac operon when glucose and lactose are present?

Answer 7

lac operon expression is off

Question 8

What happens to the lac operon when glucose is absent and lactose is present?

Answer 8

lac operon expression is on

Question 9

What happens to the lac operon when glucose is present and lactose is absent?

Answer 9

lac operon expression is off

Question 10

What happens to the lac operon when glucose and lactose are absent?

Answer 10

lac operon expression is off

Question 11

What are the stages of CAP and cAMP? When can CAP not activate the operon?

Answer 11

When glucose is low, cAMP levels are high, and cAMP binds to CAP
cAMP-CAP complex binds to the CAP binding site on the DNA which helps recruit RNA polymerase and promotes transcription
CAP cannot activate the operon if the lac repressor is already bound to the operator

Question 12

What happens to the lac operon in the presence of lactose?

Answer 12

When lactose is present it is converted to allolactose which binds to the repressor causing a conformational change in the repressor to make it unable to bind to the operator, and with the repressor removed, RNA polymerase can bind to the promoter and begin transcribing lacZ, lacY, and lacA genes

Question 13

Why is the lac operon expressed when lactose is present and glucose is absent?

Answer 13

because lactose removes the repressor and low glucose allows CAP to bind to the promoter

Question 14

What are transcriptional regulators?

Answer 14

modular proteins with domains with distinct functions

Question 15

What do transcriptional activators do?

Answer 15

increase the frequency of initiation

Question 16

What do transcriptional repressors do?

Answer 16

decrease the frequency of initiation

Question 17

How can regulatory proteins, like cAMP-CAP complex, find their specific DNA target sites with enhanced speed and accuracy compared to simple 3D diffusion alone?

Answer 17

Facilitated diffusion

Question 18

How do CAP and lac repressor bind to DNA?

Answer 18

Helix-turn-helix motif… by fitting into the major groove

Question 19

What is the helix turn helix motif?

Answer 19

2 alpha helices connected by a short turn, where the second helix (recognition helix) makes specific contacts with the DNA bases to ensure accurate binding and the first helix helps stabilize this interaction

Question 20

What does the recognition helix do?

Answer 20

recognizes a specific DNA sequence by inserting into the major groove and identifies the sequence through non covalent interactions between amino acid side chains and exposed base pairs in the groove

Question 21

What happens once CAP binds to cAMP?

Answer 21

1. conformational change in CAP
2. protein can now bind to specific DNA sequences located near promoters
3. cAMP-CAP complex interacts with RNAP, stabilizing RNAP binding to the promoter and increasing transcription rate
4. gene regulation allowing bacteria to adapt to their environment
5. binding of cAMP-CAP complex to the DNA can also cause the DNA to bend which helps facilitate the interaction between the complex and RNAP

Question 22

What is DBD and what does it do?

Answer 22

DNA binding domain… the part of the lac repressor protein that binds to the operator DNA sequence and preventing the transcription of the lac operon genes

Question 23

How does the lac repressor convert from a non-specific DNA binding state to a specific complex?

Answer 23

binding to the lac operator

Question 24

What is DNA looping? What is a classic example?

Answer 24

3D folding of a DNA molecule where distant regions of the DNA come into close contact, often facilitated by proteins binding to specific DNA sequences
arabinose operon

Question 25

What is the Trp operon? When does it produce tryptophan?

Answer 25

set of genes in prokaryotes, like bacteria, that work together to synthesize the amino acid tryptophan regulated to only produce tryptophan when it's needed using 2 key mechanisms

Question 26

What is the attenuation mechanism of the trp operon?

Answer 26

pausing and termination of the ribosome on the nascent mRNA during transcription

Question 27

What is the repression mechanism of the trp operon?

Answer 27

where excess tryptophan activates a repressor protein to block transcription

Question 28

What happens to the trp operon during low tryptophan?

Answer 28

trp operon is turned on because the repressor protein is inactive and can't bind to the operator which allows RNAP to initiate transcription of genes needed for tryptophan synthesis

Question 29

What happens to the trp operon when during high tryptophan?

Answer 29

trp operon is repressed meaning the transcription of the tryptophan synthesis genes is shut down... high levels of tryptophan cause amino acid to bind to the trp repressor protein, activates the represor protein, which then binds to the operator sequence of the trp operon, then RNA polymerase is blocked from accessing the promoter and transcribing genes necessary for tryptophan synthesis

Question 30

How does tryptophan act as a corepressor?

Answer 30

binding to the trp repressor protein and activating it to bind to the operator which physically blocks RNA polymerase from transcribing the operon, preventing the cell from making more tryptophan

Question 31

What are riboswitches?

Answer 31

RNA sequences that control gene expression by changing their 3D structure in response to a specific small molecule or metabolite, like a vitamin or ion

Question 32

What is ORF?

Answer 32

open reading frame... the coding sequence of the gene whose expression is being regulated... a riboswitch located in the noncoding part of the mRNA senses a specific molecule then modifies its own structure to control whether the downstream of ORF is successfully translated into a protein

Question 33

How is transcription termination regulated?

Answer 33

either by promoting the formation of a hairpin structure that causes the RNAP to stall and terminate or by preventing the formation of the termination structure

Question 34

What does low concentrations of purine compounds mean?

Answer 34

the metabolite binding region of the riboswitch (aptamer) remains unbound allowing the mRNA to fold into a structure that promotes the transcription of genes needed for purine production purine biosynthesis is to be on

Question 35

What does high concentrations of purine molecules mean?

Answer 35

excess purine acts as a ligand binding to specific regions of the mRNA which triggers a conformational change in the mRNA then halting genes expression purine biosynthesis is to be off

Question 36

What is the Shine-Dalgarno ribosome binding sequence?

Answer 36

acts as a ribosome binding site that keeps a gene on by allowing the ribosome to bind to the mRNA and correctly align with the start codon

Question 37

What does the SD sequester do?

Answer 37

sequestration of the SD sequence turns a gene off by blocking the ribosomes ability to bind to the mRNA which prevents the initiation of protein synthesis

Question 38

How does Rho function in the riboswitch control of termination?

Answer 38

Rho functions as a termination factor whose activity is regulated by the riboswitches conformational state... when a ligan binds to the riboswitch it causes a conformational change that either sequesters Rut sites (to prevent Rho from binding) or exposes them (allowing Rho to bind to the nascent RNA)

Question 39

What is the magnesium transporter in the riboswitch control of termination? High vs low magnesium levels?

Answer 39

the gene product whose expression is regulated by the riboswitch high magnesium: ions bind to the riboswitche's aptamer domain and the folded structure causes RNA polymerase to halt transcription prematurely low magnesium: riboswitch aptamer does not bind metal ions and RNA adopts unfolded conformation without the terminator structure and so RNAP proceeds along the gene