Gene Regulation

Question 1

Briefly outline how gene regulation is started

Answer 1

1. RNA polymerase must find the promoter sequence in DNA before it can start transcribing . The polymerase can bind the DNA directly (in bacteria)

Or

2. Seek out transcription factors that bind to the promoter (in eukaryotes)

Question 2

Outline general transcription in bacteria

Answer 2

General transcription in bacteria occurs by binding of RNA polymerase to the promoter region of a gene, the subsequent unwinding of the DNA double helix to reveal a single stranded DNA template, and the synthesis of an RNA transcript

Question 3

Contrast regulated and constitutive

Answer 3

Constitutive
-Some genes (constitutive genes) are always expressed within a cell

-Constitutive genes are often refferred to as “housekeeping” gene

Regulated
-Other genes only need to be expressed at certain times

-Therefore, to reduce wasted effort, many genes are regulated, and only expressed under certain conditions

Question 4

How is gene expression regulated?

Answer 4

This regulation can occur at many differentiated steps of gene expression, including:

• Transcription
• mRNA processing
• translation
• Regulation of protein half life

However, the majority of regulation is in bacteria takes place at the transcriptional level

Question 5

What is an operator in prokaryotes?

Answer 5

In bacteria, regulated genes have an upstream region adjacent to promoter called the operator

The operator is a sequence of DNA which is a binding site for specific proteins that help to regulate gene expression

Question 6

What are repressors and activators?

Answer 6

There are two main types of regulatory protein in bacteria:

1. Repressors: bind to an operator region and prevent RNA polymerase from initiating transcription
2. Activators bind to or near an operator region and allow RNA polymerase to initiate transcription

A given gene (or group of genes ) may use either or both types of protein for regulation

Question 7

Who discovered the lac Operon?

Answer 7

Jacob and Mood studied the regulation of genes required for the metabolism of lactose in bacteria (coordinate regulation)

Won the Nobel prize in physiology or medicine, 1965

Question 8

What is the responsibility of each gene in the Lac operon?

Answer 8

LacZ- B-galactosidase
(Lactose> glucose + galactose)
(Lactose > allolactose)

LacY- permease

(Active transport of lactose across cell membrane )

LacA-Transacetylase
(Galactose > acetylgalactose)

Question 9

Outline the map of the Lac Operon

Answer 9

All three genes share the same promoter (lacP), the same operator (lacO) and are transcribed as a single mRNA(polycistronic )

The gene for a regulatory protein (lacI) is found nearby, but separately from the Operon

Question 10

Explain Lac operon gene regulation

Answer 10

Since the role of Lac is to break down the sugar lactose, the structural (protein coding) genes lacZ, lacY and lacA should only to be expresssed when lactose is present in the cell

-lacI gene encodes a repressor protein that shuts the system down when lactose is not present

Question 11

Why is the Lac operon inducible ?

Answer 11

• If lactose is added to a cell, the Lac operon will be ‘turned on’
• Therefore, we say that the system is inducible

Question 12

Why is the Lac operon inducible?

Answer 12

Repressor (LacI) bound to operator, RNA polymerase cannot initiate transcription at the promoter

No lactose- system turned OFF

Question 13

How is lactose induced?

Answer 13

If lactose is added, there will be formation of the isomer, allolactose, which will bind to the repressor

The allolactose-bound repressor undergoes a conformational change and dissociates from the operator sequence. RNA polymerase is then free to initiate transcription.

Lactose present- system turned ON

Question 14

What are the effects of structural genes of Lac operon having mutations?

Answer 14

LacZ-, LacY-, LacA-: structural gene mutations lead to non-functional proteins

Question 15

What is the effect of lacP- mutations?

Answer 15

Non-functional promoter, RNA pol cannot bind so genes will not be expressed

Question 16

What is the effect of lacO- mutations?

Answer 16

Non-functional operator, repressor cannot bind.

Since the system cannot be shut off, this is a constitutive mutation

Question 17

What is the effect of lacI- mutation ?

Answer 17

Non-functional repressor, unable to bind the operator to shut off transcription

Question 18

What the effect of lacI^S mutations?

Answer 18

Super-repressor, unable to dissociate from operator. System always off

Question 19

Contrast Lac operon positive and negative control

Answer 19

When a repressor is used to turn the system off, that system is under negative control. The LacI repressor is an example of this

The Lac operon is also under positive control, where an activator protein is used to increase the efficiency of transcription

Question 20

E. coli’s favorite carbon source is glucose…

Answer 20

• While E. coli can metabolize lactose with the products of the Lac operon, it actually ‘prefers’ glucose as a carbon source
• Therefore, when glucose is present, there is no need to express the genes in Lac operon

Question 21

Summarize positive control of Lac operon

Answer 21

• Genetic expression of Lac operon occurs only if a regulator molecule directly stimulates RNA production (that molecule is cyclic AMP)
• If a cell runs out of glucose, a small molecule (cyclic AMP or cAMP for short) is produced by active Adenylyl cyclase (converts ATP to cAMP)

This molecule is a ‘hunger signal’ that permits the expression of genes that break down other sugars, including lactose

-cAMP binds the activator protein CRP( cAMP receptor protein) or CAP (catabolite activator protein), which can then bind lacP to help activate transcription

Question 22

Explain the effect of the hunger signal in the Lac operon

Answer 22

Unless the hunger signal is given, Lac operon is not expressed even in the presence of lactose

Inactive CRP
no cAMP around means that gluc9se is in the environment

When glucose is present, no cAMP is produced so CRP is inactive

RNA pol cannot EFFICIENTLY initiate transcription

The hunger signal, in the presence of lactose

If no glucose is present, cAMP is produced, which binds to and activates CRP

Activated CRP can then bind to the promoter

Question 23

Where do s allolactose become bound during Lac operon expression?

Answer 23

Bound to the repressor so lactose is in the environment

Question 24

What happens after CRP binds to the promoter?

Answer 24

When activated CRP binds to the promoter, RNA pol can enter and initiate transcription (IF lactose is present)

If the hunger signal is given, but lactose is absent, the Lac operon isn’t expressed

Repressor bound to operator (lactose absent)—> blocks RNA pol from transcribing the operon

Question 25

Summarize the Lac operon

Answer 25

The Lac operon will only be transcribed when lactose is present (repressor unbound the operator) and glucose is absent (CAP/cAMP is bound to CAP binding site)

Question 26

In what circumstances is the Lac operon on?

Answer 26

Glucose absent, lactose present No repressor bound, activator bound - it’s ON All other circumstances, it’s off

Question 27

Give an overview of gene regulation in eukaryotes

Answer 27

Regulation of gene expression is a complex, multi-layered process that is crucial to correctly drive and maintain cell identity during development and adult life All of these cells contain the exact same genetic information Different expression of the genes, I.e. Gene regulation results in this variety of cell types. Thus, regulation is more complex in eukaryotes than in bacteria

Question 28

Why is transcriptional control more complex in eukaryotes?

Answer 28

Because of nuclear compartmentalization, chromatin remodeling, recruitment of transcription machinery, transcription initiation, elongation, termination

Question 29

What are the key concepts of Cis elements?

Answer 29

DNA sites where proteins and trans regulatory elements will bind - Latin root, means same side as - DNA motifs which are binding sites for transcription factors enhancer protein binding sites - These are the DNA sequence itself, the consensus sites mentioned when we discuss transcription

Question 30

What are the key concepts of trans regulatory elements?

Answer 30

- Latin roots which means across or opposite side - These proteins are encoded for by different genes in the genome - These are typically proteins such as transcription factors/ enhancer binding proteins - May have positive or negative effects

Question 31

Where are trans acting elements synthesized?

Answer 31

Synthesized from genes that are different from the genes targeted from regulation. Trans-acting factors bind to cis-acting elements of DNA

Question 32

Explain Cis elements

Answer 32

May be classsified by location and function Typically they have a consensus sequence Sequence among many genes isn’t exact, it is similar Each of these is a Cis regulatory element: 1. Basal promoter sequence These bind the general transcription factor proteins which are associated with RNA Pol 2. Proximal control regions These bind transcription factor proteins, and are found near the promoter 3. Enhancer sequence Are found far away from the promoter

Question 33

Why bother with basal and upstream promoters?

Answer 33

Multiple levels of control allow for tight regulation

Question 34

What are the regulatory elements?

Answer 34

Distal- enhancer element (variety of STF) Proximal - CAAT box(CTF(STF)) and GC box(Sp1)(STF)

Question 35

What are the core promoter of elements?

Answer 35

TATA Box (-25), Inr (+1), and DPE(+25) All bind to TFIID(GTF)

Question 36

Contrast Basal transcription vs enhancer elements

Answer 36

A) Basal transcription machinery allows a low level of expression B) Binding of trans factors to the proximal promoters (here CTF and Sp1) regulate frequency of transcription initiation Enhancer trans factors binding to the enhancer element mediate the response to signals such as hormones and regulate which genes are expressed at a given point in time. Mediator proteins may also bind the enhancer binding proteins +/- DNA bending can cause an enhancer element that is far from the promoter in the linear DNA molecule to interact with the transcription-initiation complex, stimulating transcription

Question 37

What are the enhancer regions?

Answer 37

Bending (or looping) of DNA allows distant regions to contact physically -Enhancer regions can be located before or after a gene (or even in in an intron) - Enhancers can be quite from the gene (thousands, or many thousands of base pairs) - May be more then 200 bp away and still function as an enhancer

Question 38

Where can enhancer regions be found?

Answer 38

Can be found upstream from the promoter region - Thousands of base pairs can separate the enhancer sequence from the gene it regulates - An enhancer sequence can be downstream from the promoter region

Question 39

What is the DNA Binding domain?

Answer 39

Transcription factors have DNA binding domains that only bind to certain DNA sequences

Question 40

What is the dimerization domain?

Answer 40

Two transcription factors bind together to form a functional DNA binding unit called a dimer. Formation of a dimer adds an extra element of complexity and versatility

Question 41

What is the activation/repression domain?

Answer 41

Binds to the enhancer binding proteins or other transcription factors and modulates their function

Question 42

What are the parts of a transcription factor?

Answer 42

- Activation and/or repression domain - dimerization domain - DNA binding domain

Question 43

What are 3 examples of eukaryotic transcription factors that must dimerization to function?

Answer 43

Zinc finger Helix-turn-helix Leucine zipper

Question 44

Explain how different genes can possess the same response element

Answer 44

- Provides a mechanism to COORDINATE gene regulation - Allows multiple genes to be regulated together - Allow different genes to be turned on and off at the same time - Stage specific expression during embryo development - Tissue specific expression - Allow response to external stimuli-Many examples… including: - hypoxia (hypoxia response element) - hormonal (hormonal response element) - stress and heat (heat shock element)