Gene Regulation (Exam 4 Part 1)

How do we get different cell types in our bodies?

The cells of an organism have the same DNA. Control of gene expression (turning genes ON and OFF) give cells their various properties

Gene expression

The process by which information from a gene directs the synthesis of a gene product in the form of a protein or a functional RNA molecule. This ultimately affects a phenotype of the cell/organism.

Scientists attempted to describe the fact that different cells became specialized and perform different functions even though they started from the same original cell by

hypothesizing how cells became differentiated

The two hypotheses on how cells became differentiatied

Certain pieces of DNA were lost from cells so they only retained the DNA that made that cell type

Parts of the DNA were turned on or off so that only the right genes were expressed.

The correct hypothesis

Cells retain all the instructions to make a new organism

Sir John Gurdon demonstrated that

putting the nucleus (genetic information) from an adult, differentiated frog cell into an egg cell with the nucleus removed could become a whole organism. The implication of this work was that genes had to be turned on and off in different cell types

Cells use exquisite control at almost every level to

regulate the amount and types of protein produced by a cell

Initiation of transcription is the main point of

control

Promoters are

only part of the story

Additional DNA elements called regulatory sequences bind

proteins, which control gene expression

• In prokaryotes: these are called operators

• In eukaryotes: these are called enhancers and proximal promoter regions

Operators are in

prokaryotes

Enhancers and proximal promoters are in

eukaryotes

THe proteins that bind regulatory elements are called

transcription regulators (may also be called transcription factors NOTE THIS IS DIFFERENT FROM GENERAL TRANSCRIPTION FACTORS)

Transcription regulator proteins interact with DNA through a

DNA bidning domain

Transcription regulator (proteins) interact with DNA through

contacts in the major and minor groove

Amino acid side chains make

hydrogen bonds (ionic and hydrophobic too!) with nucleotide base pairs in the major (and minor) groove

Each of the four base pairs have unique patterns of

hydrogen bond donors and acceptors so the transcription regulator can recognize a specific sequence in the DNA

Transcription regulators can tell a

T-A pair from an A-T base pair and a G-C from a C_G base pair chemically — they can “read” the DNA!

Transcription regulators bind DNA at

specific sequences

Transcription factor binding site (often called a response element or motif):

short DNA sequence that is recognized and bound by a transcription factor to regulate gene transcription

Consensus Motif:

The common or “average” DNA sequence shared among many binding sites for the same transcription factor regulator

How are regulatory DNA sequences usually written?

They are written as the preferred nucleotide at each position in the DNA sequence, from 5’ → 3’.

In a sequence logo, what does letter height represent?

The height of each letter shows how strongly that nucleotide is preferred at that position. Larger letters = stronger preference.

What happens if a DNA sequence closely matches the preferred binding sequence?

The regulatory protein is more likely to bind strongly to that DNA sequence.

With sequenced genomes we can

scan for these sequences and predict which genes will be regulated by the regulatory factor

What are transcription factors and what do they do?

Transcription factors are proteins encoded by genes that bind specific DNA sequences and regulate transcription by helping or blocking RNA polymerase at the promoter.

How do transcription factors recognize specific DNA sequences?

Amino acids in the protein form chemical bonds with nucleotide bases in the DNA, allowing sequence-specific binding.

How are transcription factors different from general transcription factors (GTFs)?

Transcription factors regulate specific genes, while GTFs are generally required for transcription initiation.

Th role of transcription factors is to

promote or inhibit transcription by affecting how RNA polymerase interacts with the promoter

Transcription regulators ____ transcription

inhibit (repress) or promote (activate)

Many bacterial mRNAs are

polycistronic

When might you like to express multiple gens at the same time?

in response to changes in the environment

Many bacterial genes are organized into

operons

Operon

Cluster of genes that are co regulated

Operator

Regulatory DNA sequence in/near the promoter region (distinct from the -35 and -10 sequences)

Repressor prevents RNA polymerase from

initiating transcription

Activator promotes RNA Polymerase

transcription initiation

In the presence of tryptophan (lots of it) would you expect a repressor or activator of the operon to be active?

No because High tryptophan → active repressor → operon OFF

• Low tryptophan → inactive repressor → operon ON

E. coli cells control the tryptophan operon by

sensing the concentration of tryptophan in hte cell

Trp repressor is an example of an

allosteric enzyme

• binding of an effector changes the shape of the protein

• In this case binding of tryptophan permits DNA binding

Example of feedback inhbition

Tryptophan binding changes the

conformation of the repressor — opens the structure to permit DNA binding

Lac Operon is the

regulation of enzymes that breakdown lactose in the bacteria

LacZ

breaks down lactose to make glucose

LacY

allows lactose to enter cell

LacA

promotes lactose breakdown

Glucose is the preferred food source for

E. coli

LacI bidn the lacO operator in the absence of lactose to

physically block RNA polymerase from binding

In the presence of lactose, allolactose binds

Lacl to inhibit its DNA binding ability, when this bind happens it causes the repressor to dissociate from the DNA (ie fall off) allowing transcription of the operon

When glucose levels are low, cAMP levels are

high, promoting CAP activator binding to the DN

If there is a lote of lactose and no glucose then

the cell wants a lot of expression, activator promotes RNA polymerase transcription initiation

Combinatorial Control

Regulatory elements act like “and” “or” and “not” gates to control gene expression

All steps can be regulated but the main site of control is

transcription initiation

Eukaryotic transcription regulators can act far from the

promoter

What are enhancers?

Enhancers are DNA regions where transcription regulators (activators) bind to increase transcription.

How far away can enhancers be from the start of transcription?

They can be thousands of base pairs away from the transcription start site.

What do activators do?

Activators help form the transcription initiation complex (pre-initiation complex, PIC) by promoting the binding of general transcription factors and RNA polymerase II.

Regulator DNA sequences are sequences outside of the core promoter that

control when and how much transcription occurs

Repressors

• Physically block activators or GTF/RNA Pol II by binding

• DNA Bind activators to prevent their DNA binding

• In some way prevent initiation

Activators increase

ability of mediator, general transcription factors (GTF) and RNA Polymerase II to bind to the promoter

Binding site for the same transcription regulator can be found in

the promoters/enhancers of many genes, these genes can be anywhere in the genome they do not need to be near each other

The coding sequence can be on

either strand of DNA. By convention we illustrate the coding strand

How do we get different cell types in our bodies?

Differential gene expression

Characteristics of stem cells

• Unspecialized

• Potent (able to become different cell types)

• Self-renew

Types of stem cells

• Embryonic

• Adult

• Induced Pluripotent (iPSC)

What is Waddington’s Landscape?

A model that shows how cells become more specialized over time by moving down a “hill” of developmental potential, with gene expression guiding their fate.

What does the “top of the hill” represent in Waddington’s Landscape?

A cell with maximum developmental potential (zygote), which can become many different cell types.

What happens as a cell moves down Waddington’s Landscape?

It becomes more specialized, loses developmental potential, and commits to a specific cell fate.

What drives differences between cell types?

Changes in gene expression.

What is a totipotent cell?

A cell (like a zygote) that can form all cell types in the organism, including extra-embryonic tissues like the placenta.

What is a pluripotent cell?

An embryonic stem cell that can become almost any body cell type but not extra-embryonic tissues.

What is a multipotent cell?

An adult stem cell that can become a limited range of related cell types.

What is a unipotent cell?

A fully differentiated cell that can typically only produce one cell type.

Why is it hard for cells to “go back uphill” in Waddington’s Landscape?

Because gene expression patterns become stabilized, locking in cell identity.S

Some transcription factors are

“Master regulators: that drive expression of many genes. These genes (and the genes they control) dramatically alter the cell’s fate and function

Transcription factors that are master regulators can bind

their own promoters and each other’s promoters in a positive feedback loop

Many genes require a combination of

regulators to turn on expression

The cell integrates information (AND, OR, NOT) to determine if

a gene should be ON or OFF in a particular cell

Combinatorial Control:

Groups of transcription regulators work together to ensure the proper set of genes are turned ON (or OFF) altering cell fate and function

From a limited number of transcription regulators,

you can get many different cell types

Combinations of transcription regulatory factors can drive

differentiation of different cell types

Sir John Gurdon demonstrated that putting the nucleus (genetic information) from a differentiated frog cell into an egg cell with the nucleus removed could

become a whole organism

All the information needed to create a new organism is found in their

DNA

A lab demonstrated that isolated adult skin cells could be

pushed back in developmental time to an embryonic like cell state. These cells are called induced pluripotent stem cells or iPSCs.

Expression of three transcription factors could

reprogram adult cells into stem cell-like cells. oct4, sox2, and klf4 known as the Yamanaka factors

The expression of master regulators can revert an

adult cell back to a stem cell like fate, huge implications for medical research and treatmetns

What is epigenetics?

Changes in gene expression caused by chemical modifications to DNA or DNA-associated proteins, without changing the DNA sequence itself.

Why is epigenetics described as “above” the genetic code?

Because it affects how genes are read and expressed, not the actual DNA sequence.

How do epigenetic changes affect gene expression?

They change how accessible or readable DNA is, which can turn genes on or off.

When do epigenetic changes occur?

During development and in response to environmental factors.

What is an epigenetic profile?

The unique pattern of epigenetic marks that helps define a cell’s identity and specialization.

Can epigenetic changes be passed on during cell division?

Yes, they can be inherited by daughter cells when cells divide.

Why is epigenetics important for adaptation?

Because it is reversible, allowing organisms to adjust gene expression in response to the environment.

What is DNA methylation?

The addition of methyl groups to DNA, often reducing gene expression. Occurs on certain cytosine bases (usually found in CpG pairs). tends to turn OFF genes in the area by recruiting in proteins that block transcription

What is histone acetylation?

The addition of acetyl groups to histone proteins, usually increasing gene expression by loosening DNA.

What is histone methylation?

The addition of methyl groups to histones, which can either increase or decrease gene expression depending on context.

What are chromatin remodeling factors?

Proteins that reposition or restructure chromatin to control how accessible DNA is for transcription.

Types of modifications

DNA methylation, histone acetylation and methylation, chromatin remodeling factors

DNA accessibility controls

what genes are expressed

Common theme in biology: Alter the accessibility of

DNA, RNA, or protein elements to control their function