Chapter 15 - Regulation

What do the mechanisms of gene regulation, or genetic control, determine?

where, when, and how much a gene is expressed

every gene is a ________ (transcribed region) of DNA

RNA-coding region

What are transcription factors?

DNA-binding proteins that recognize specific sequences within the regulatory regions near the gene to either activate or repress transcription

operon

a cluster of structural genes with related functions under the control of a common regulatory system that can respond to changes in environmental conditions

• common in prokaryotes but rare in eukaryotes

The products of the structural genes of the lac operon in E. coli are involved in what?

utilization of lactose for energy

lac operon: these genes will only be expressed if _____ is available; but also only if the cell needs to use lactose for energy

lactose

When are the genes of lac operon expressed?

only if glucose is absent and lactose is available

lacZ gene product

B-galactosidase

What is B-galactosidase?

an enzyme that breaks down lactose (a disaccharide) into galactose and glucose

• can also isomerize lactose into allolactase

lacY gene product

permease

What is permease?

a membrane transporter for lactose and facilitates the entry of lactose into the bacterial cell

lacA gene product

transacetylase

What is transacetylase?

not involved in lactose metabolism and involved in the removal of by-products of lactose digestion from the cell

structural genes under operon control

lacZ, lacY, and lacA

control regions

lacP (promoter or P_lac)

lacO (lac operator)

lacP

binding site for RNA polymerase

lacO

binding site for the regulator protein; it overlaps lacP

regulator locus

lacI

P_i

lac(I)

the gene that codes for the regulator protein

P_i

the promoter for the lacI gene

What happens in the absence of lactose?

the regulator protein (repressor) binds to the operator, blocking RNA polymerase from binding to the promoter

• repression of the structural genes, but this system is leaky

What is the operon inducer?

allolactose which binds to the repressor protein and inactivates it

• this activates the expression of the structural genes

if glucose is available, the lac structural genes are ___, even if lactose is present

off

When does the binding of RNA polymerase to the promoter occur?

only if glucose is absent

adenylate cyclase

enzyme that catalyzes the hydrolysis of ATP into cAMP+PP is induced when glucose is absent

• cytoplasmic levels of cAMP (catabolite) increase

What does cAMP activate?

catabolite activated protein (CAP)

the activated CAP binds to a site next to the _____ and facilitates the binding of RNA polymerase to it

lac promoter

What is cAMP?

catabolite that binds to the catabolite activated protein (CAP) to activate it

What is the catabolite activated protein (CAP) necessary for?

stable binding of RNA polymerase to the lac promoter

• only active when glucose is absent

What happens in the presence of glucose?

cAMP levels decrease and CAP remains inactive

• RNA polymerase does not bind the promoter efficiently

• the operon is OFF (no expression of the structural genes)

What are the products of the five structural genes of trp operon?

enzymes involved in the biosynthesis of the amino acid tryptophan (Trp)

if Trp is absent

structural genes are expressed

if Trp is present

structural genes are turned off by Trp itself

promoter (trpP)

binding site for RNA polymerase

operator (trpO)

binding site for the repressor protein

repressor gene (trpR)

codes for the repressor protein (repressor gene promoter P_R)

Trp absent

the repressor protein remains inactive

• RNA polymerase binds to the promoter and initiates the transcription of the structural genes

Trp present

Trp binds to the repressor protein and activates it. The binding of the Trp-activated repressor to the operator prevents the binding of RNA polymerase to the promoter.

EMSA (electrophoretic mobility shift assay)

a method used for detecting specific DNA-protein interactions, such as the specific binding of transcription factors and other regulatory proteins to regulatory regions of the chromosome

example of EMSA

establishing the specific binding of the trip repressor protein to the trp operator (trpO) in the presence of tryptophan

mix in a test tube:

• a fragment of DNA containing trpO

• E.coli cell extract (contains all cellular proteins)

• tryptophan (Trp)

• specific antibodies against the trp repressor and other cellular proteins, such as the lac repressor

five experiments

1. DNA alone

2. DNA + cell extract (no Trp)

3. DNA + cell extract + Trp

4. DNA + cell extract + Trp + anti-lac repressor antibody

5. DNA + cell extract + Trp + anti-trp repressor antibody

in the five experiments, what to do?

• slow gel electrophoresis of samples from the contents of each tube

• electrophoretic mobility shifts occur due to the different sizes of the complexes that form

RNA polymerase I

the larger rRNA genes (5.8s, 18s, 28s)

What transcribes the eukaryotic genes?

RNA polymerases

RNA polymerase II

all the protein-coding genes

RNA polymerase III

the small rRNA (5s) gene and all the tRNA genes

What are some small nuclear RNAS (snRNAS) and small cytoplasmic RNAs (scRNAs) transcribed by?

RNA polymerase II and others by RNA polymerase III

heterochromatin

transcriptionally inert due to the tight association of DNA with histones, which prevents RNA polymerase from binding to gene promoters

Why is the ability of the cell to alter the association of DNA with histones essential?

to allow gene regulatory proteins and RNA polymerases to bind to DNA

What does histone acetylation weaken?

the interaction between basic histones and the acidic DNA molecule, causing chromatin decondensation

What does not bind to DNA but regulate histone acetylation?

HATs and HDACs

What doe positively charged tails of nucleosomal histone proteins interact with?

negatively charged phosphate groups of DNA

Acetylation of the tails weakens their interaction with what?

DNA and may permit some transcription factors to bind to DNA

What do transcription activators recruit?

histone acetyl transferases (HATs), causing chromatin decondensation

• act as coactivators but do not bind to DNA

What do transcription repressors recruit?

histone deacetylases (HDACs) causing chromatin condensation

• act as corepressors but do not bind to DNA

What does the flowering locus C (FLC) code for?

a transcription factor that represses flowering but is only expressed if the histones on the locus are acetylated

What is the gene product of flowering locus D (FLD)?

a histone deacetylase that specifically inactivates the FLC to allow flowering

Arabidopsis thaliana (Thale cress)

Most widely studied plant in biology. It has contributed our understanding of the genetic, molecular, cellular, and develop-mental biology of flowering plants. It is as important to botany as Drosophila is to animal genetics

What do acetyl groups on histone proteins destabilize?

chromatin structure

What does the term transcription factor refer to?

any transcription regulator protein that binds to a specific DNA sequence

What are cis-acting elements?

DNA sequences that are necessary for the control of transcription (the regulatory regions of chromosome): promoters, enhancers, and silencers

What are trans-acting factors?

proteins that are necessary for the control of transcription: the transcription factors that bind to the cis-acting elements

promoters

the binding sites for the RNA polymerase II transcription initiation complex

• a key component of many eukaryotic promoters is a TATAAA sequence within them known as the TATA box

enhancers

binding sites for transcriptional activator proteins

silencers

binding sites for transcriptional repressor proteins

What are enhancers required for?

stimulated transcription

DNA sequences for enhancers vary widely and are recognized by what?

a large variety of transcription activators

What will the activation of a gene in any particular cell depend on?

whether the cell has the right activators to bind to the gene enhancers

What happens by facilitating up-regulation?

enhancers determine where, when, and how much transcription occurs

promoter alone

only the basal transcription factors may bind to DNA, and only the basal transcription apparatus may form

• only very low or undetectable transcription can occur

With the right enchancers

transcriptional activators bind to enhancers

• stimulated transcription (biologically significant)

What positions can enhancers be?

distant upstream, downstream, or inverted positioin

• still be effective in stimulating transcription (CANNOT MOVE PROMOTER)

Insulators (boundary elements)

DNA sequences that block the effect of enhancers in a position-dependent manner when insulator-binding proteins are bound to them

• This prevents enhancers from stimulating the transcription of the wrong genes on the same chromosome

What is the purpose of the RNA polymerase II transcription initiation complex?

to direct RNA polymerase II to the correct place on the promoter behind the gene transcription initiation site (+1), because the polymerase itself does not recognize any specific DNA sequences

TFIIs

transcription factors of RNA polymerase II

TFIID

transcription factor D of RNA polymerase II

• this recognizes the TATA box and binds to it

What do transcriptional activators bind to?

upstream and in some cases downstream sequence elements (enhancers) which are necessary for stimulated, biologically significant, levels of transcription

RNA polymerase II transcription initiation complex (basal transcription apparatus)

contains TFIID, other TFIIs, and RNA polymerase II. DNA loops to allow transcriptional activators, bound to enhancers, to interact with the complex

Why does DNA loops?

to allow transcriptional activators, bound to enhancers, to interact with the complex

What are the products of the GAL structural genes in yeast?

proteins that metabolize the sugar galactose

What does the transcription of GAL structural genes requires?

enhancer UAS_G (upstream activating sequence of GAL genes) which is permanently occupied by the transcription activator GAL4

What happens in the absence of galactose?

the GAL80 protein binds to GAL4 and prevents it from activating the transcription of the GAL structural genes

What happens when galactose is available?

it activates the GAL3 protein, which interacts with GAL80 to displace it and expose the GAL4 trans-activating domain

What is required that interacts with GAL4 and the transcription initiation complex?

mediator protein

Barbara McClintock’s transposition model (1950)

The expression of a gene that is necessary for the synthesis of the pigment anthocyanin can be suppressed by a silencer that she named Dissociator. It is a transposon: a segment of DNA that can move from one location on a chromosome to another. If Dissociator is transpositioned, the suppression of the pigmentation gene is released, and anthocyanin can be made.

How long the Dissociator stays next to the pigmentation gene before “jumping” affects the degree of pigmentation and mottling in a maize kernel. The reddish-purple patterns caused by the transposon may be dots, blotches, or streaks. If the transposon stays next to the pigmentation gene long enough, the grain will be completely unpigmented.