Lecture 15: Control of Gene Expression I: Transcriptional Control

Question 1

What is a repressible operon?

Answer 1

When an excess of product leads to a shutdown of the production of enzymes that synthesize that product

Question 2

In a repressive operon, what does the repressor need in order to bind to the operator and block RNA polymerase from binding?

Answer 2

A co-repressor (in the Trp operon, it’s tryptophan, which is the product)

Question 3

When is the Trp operon on and when is it off?

Answer 3

On when tryptophan is low (repressors don’t have their co-repressor so can’t bind operator), and off when tryptophan is high (lots of tryptophan to be the co-repressor)

Question 4

What is signal transduction?

Answer 4

Mechanisms that allow a cell to detect internal or external changes and respond appropriately

Question 5

Components of the two-component signal transduction system

Answer 5

Sensor and response regulator

Question 6

Purpose of sensor in two-component signal transduction system

Answer 6

Phosphate transferred to histidine residue in “transmitter domain” as response to environmental change

Question 7

Purpose of response regulator in two-component signal transduction system

Answer 7

“Receiver domain” that receives phosphate from sensor, becoming active (can either activate or suppress genes)

Question 8

Sensor in PhoR-PhoB system and how it works

Answer 8

PhoR, a transmembrane protein: inactive when phosphate bound, active when phosphorylated (Pi does not remain bound)

Question 9

PhoR-PhoB system: what does activated PhoR do?

Answer 9

Phosphorylated PhoB, the response regulator, which then becomes a transcription factor to turn on genes that will help cope with low phosphate levels

Question 10

What is the preferred carbon source for energy in bacteria like E.coli?

Answer 10

Glucose because it’s easier to use; they only use lactose when glucose is absent

Question 11

Enzymes required for lactose metabolism in E.coli (lac operon)

Answer 11

Per ease to transport lactose into cell, and β-galactosidase to cleave lactose molecule to yield glucose and galactose

Question 12

Which protein does lacZ gene make?

Answer 12

β-galactosidase

Question 13

Which protein does lacY make?

Answer 13

lactose permease

Question 14

Which protein does lacA make?

Answer 14

a transacetylase

Question 15

Which protein does lacI make?

Answer 15

Repressors protein that binds to operator on DNA (made all the time, as it turns of the other lac genes since they’re usually not needed)

Question 16

Control mechanisms for lac operon

Answer 16

lac repressors (negative control) and CAP-cAMP complex (positive control)

Question 17

Recognition sites of lac repressors (lacI)

Answer 17

Site that recognizes specific operator sequence for lac operon, and an allosteric site that can bind lactose and lactose analogs like IPTG

Question 18

What happens when lactose or IPTG binds lacI?

Answer 18

Repressor is INACTIVE (off) which means the lac genes are ON

Question 19

Lac operon: what happens when lactose is absent?

Answer 19

Repressors binds operator region, blocking binding of RNA polymerase

Question 20

What is an inducer (how does it relate to lac operon)?

Answer 20

Inducers inactivate the repressors, leading to expression of lac genes (in the lac operon, lactose and IPTG are inducers)

Question 21

Methods of transcriptional control in prokaryotes

Answer 21

Trp Operon, PhoR-PhoB, Lac Operon

Question 22

Lac operon: what happens when lacI is a constitutively active mutant (I-)?

Answer 22

All lac genes synthesized at all times, whether or not the inducer was present

Question 23

Lac operon: where is the mutation in mutants that express lac enzymes even when repressor is present?

Answer 23

Mutation is in the operator region, blocking repressor binding (O^c for operator constitutive)

Question 24

Lac operon: what happens once glucose is gone?

Answer 24

Cells synthesize cAMP, which binds to each subunit of dimerization catabolism activator protein (CAP)

Question 25

Lac operon: what does binding of cAMP to CAP cause?

Answer 25

Conformational change, allowing protein to being to CAP site in transcriptional control region of DNA, increasing affinity of RNA polymerase for lac promoter

Question 26

Lac operon: what happens to transcription of lac genes when cAMP/CAP binds to DNA?

Answer 26

Transcription is high

Question 27

Lac operon: what happens to transcription of lac genes when glucose levels are high?

Answer 27

Transcription is low, as glucose decreases cAMP, meaning CAP doesn’t bind DNA and RNA pol has lower affinity for lac promoter

Question 28

Lac operon: is it on or off when both glucose and lactose are present?

Answer 28

Off

Question 29

How can β-galactosidase activity be assessed?

Answer 29

By cleavage of ONPG, a lactose derivative that produces a yellow compound (O-nitrophenol) once cleaved

Question 30

How can cells with the same genetic information develop into cells with different structure and function?

Answer 30

Regulation of gene expression - different cell types have different genes turned on or off

Question 31

What are housekeeping genes?

Answer 31

Common genes (like rRNA genes) that are expressed at all times in all cell types

Question 32

What does it mean the regulatory sequences (enhancers) are cis-acting elements?

Answer 32

They affect their own sequence, not neighboring ones

Question 33

What is a promoter-proximal element (PE) and where is it found?

Answer 33

Regulatory element located within 100-200 bp upstream of start site (can be cell-type specific)

Question 34

What are enhancers and where are they found?

Answer 34

Regulatory elements that can stimulate transcription (inc. of up to 1000x) from up to 50 kilometers from the start site, can be located upstream of downstream of start site (even within intron)

Question 35

How are genes inactivated in areas of chromatin condensation?

Answer 35

Histone deacetylation

Question 36

How are genes expressed in areas of chromatin decondensation?

Answer 36

Histone acetylation

Question 37

What do activators do?

Answer 37

Bind to regulatory elements (enhancers) to influence decondensation of chromatin to recruit RNA polymerase or stabilize preinitiation complex

Question 38

What do repressors do?

Answer 38

Bind to control elements (silencers) to promote chromatin condensation and stop transcription

Question 39

Parts and functions of transcriptional activators and repressors

Answer 39

DNA binding domain and activation/repression domain (interacts with other proteins or subunits)

Question 40

How do activators and enhancers stimulate transcription?

Answer 40

Activators bind to sites on enhancers, then interact with other proteins to recruit transcription machinery

Question 41

What is a mediator (related to transcriptional activation)?

Answer 41

Molecular bridge between activators and RNA Pol II; serves as a co-activator as it assists in transcriptional activation

Question 42

The correct combination of _______ ______ is needed for a specific gene to be expressed

Answer 42

Regulatory proteins

Question 43

Possible mechanisms of transcriptional activation

Answer 43

Recruit transcription factors to promoter area, stabilize pre-initiation complex, disrupt local chromatin structure, histone modification

Question 44

Histone modifications that control condensation state of chromatin

Answer 44

Acetylation of lysine, phosphorylation of serine

Question 45

What are insulators?

Answer 45

DNA sequences that prevent regulatory proteins from influencing distant genes

Question 46

What is methylated DNA often correlated with?

Answer 46

Inactive regions of the chromosomes (can be inherited during replication)