The different cell types in a multi-cellular organism differ dramatically

in both structure and function.

Cell differentiation generally depends on changes in gene

expression rather than changes in the nucleotide sequences of
the cell’s genome. Different cell types synthesize different
1
sets of proteins.
Evidence that a differentiated cell contains all the genetic instructions
necessary to direct the formation of a complete organism:

The injected donor nucleus is capable of

directing the recipient egg to produce a
normal tadpole.

2
Individual cells can regenerate an entire plant.
Dolly (5 July 1996 – 14
February 2003) was a
female domestic sheep
who was the first
mammal to be cloned
from an adult somatic
cell, using the process of
nuclear transfer.

She was cloned by Ian

Wilmut and colleagues at
the Roslin Institute in
Scotland. She lived until
the age of six.

3
 Gene Control
• Gene expression refers to • Regulation of gene
the ability of a gene to expression refers to the
produce a biologically cellular control of the
active protein. amount and timing of
changes to the appearance
of the functional product of
a gene.

Controls that act on gene expression are much more

complex in eukaryotic cells than in prokaryotic cells.
- transcriptional control vs regulation from
many different points
4
Transcription switches allow cells to respond to changes in the environment.

A cluster of bacterial genes can be transcribed from a single promoter. Each

of these five genes encodes a different enzyme; all of the enzymes are needed to
synthesize the amino acid, tryptophan. The genes are transcribed as a single
mRNA molecule, a feature that allows their expression to be coordinated. Clusters
of genes transcribed as a single mRNA molecule are common in bacteria. Each
such cluster is called an operon; expression of the tryptophan operon shown here
is controlled by a regulatory DNA sequence called the operator, situated within
the promoter.
5
 Genes can be switched on and off
with repressor proteins.

If the concentration of tryptophan inside the cell is low, RNA polymerase (blue)
binds to the promoter and transcribes the five genes of the tryptophan operon (left).

If the concentration of tryptophan is high, however, the repressor protein (dark green)
becomes active and binds to the operator (light green), where it blocks the binding of
RNA polymerase to the promoter (right). Whenever the concentration of intracellular
tryptophan drops, the repressor falls off the DNA, allowing
6
the polymerase to again transcribe the operon.
In eukaryotic cells, gene expression is regulated at
many steps in the pathway from DNA to RNA to protein.

1.Transcriptional control: when & how often a given gene is transcribed ***
2.RNA processing control: how the RNA transcript is spliced
3.RNA transport & localization control: selecting which mRNAs leave the
nucleus
4.Translational control: selecting which mRNAs are translated
5.mRNA degradation control: selecting mRNA for destabilization ***
6.Protein activity control: selecting which proteins are activated, deactivated, 7
degraded
Almost all genes — especially in eukaryotic cells — are
controlled by the combined effects of activators and
repressors.

Regulatory factors interact with general transcription factors.

– Activators
transcription factors
that act positively to
promote transcription
– Repressors
transcription factors
that act negatively to
reduce transcription
8
Activators:
• transcription factors that act positively to promote transcription
• they consist of two independent domains. The DNA-binding
domain recognizes a specific DNA sequence, and the activation
domain interacts with other components of the transcriptional
machinery.

Cooper (2000) The Cell: A Molecular Approach

The basic function of the DNA-binding domain is to anchor the transcription

factors to the proper site on DNA; the activation domain then independently
stimulates transcription by interacting with other proteins.

They act in a tissue or time dependent manner to stimulate higher levels of

transcription. 9
Enhancer sequences: A transcriptional regulatory sequence (to which
activators bind) can be located at a site distant from the promoter.

- stimulate transcription when placed either upstream or downstream of the

promoter, in either a forward or backward orientation.
- function by binding transcription factors that then regulate RNA polymerase.

This is possible because of DNA looping, which allows a transcription factor bound
to a distant enhancer to interact with RNA polymerase or general transcription
factors at the promoter.

Refer to the next slide

Cooper (2000) The Cell: A Molecular Approach

The binding of specific transcriptional regulatory proteins to enhancers is

responsible for the control of gene expression during development and
differentiation, as well as during the response of cells to hormones and
growth factors. 10
11
Enhancer sequences: A transcriptional regulatory sequence (to which
activators bind) can be located at a site distant from the promoter.

- stimulate transcription when placed either upstream or downstream of the

promoter, in either a forward or backward orientation.
- function by binding transcription factors that then regulate RNA polymerase.

This is possible because of DNA looping, which allows a transcription factor bound
to a distant enhancer to interact with RNA polymerase or general transcription
factors at the promoter.

Cooper (2000) The Cell: A Molecular Approach

The binding of specific transcriptional regulatory proteins to enhancers is

responsible for the control of gene expression during development and
differentiation, as well as during the response of cells to hormones and
growth factors. 12
Eukaryotic repressor proteins do the opposite: they decrease
transcription by preventing or sabotaging the assembly
of the initiation complex.

(A) Some repressors block the binding of activators

to regulatory sequences.

(B) Other repressors have active

repression domains that inhibit
transcription by interactions with general
transcription factors

Cooper (2000) The Cell: A Molecular Approach

Repressors inhibit the expression of tissue specific genes in

13
inappropriate cell types.
Recently discoveries indicate that noncoding RNAs (not
involved in protein synthesis) play a role in regulating gene
expression. i.e. microRNAs (miRNAs) & RNA
interference (RNAi) or silencing.

miRNAs (24-26 nucleotides) efficiently block the

production of protein that the mRNA encodes.

i.e. miRNAs control gene expression by base pairing with

specific mRNAs, essentially affecting their stability &
translation

e.g. Insulin production is subject to regulation by miRNAs.

• miRNAs target mRNAs that code for proteins involved in
insulin synthesis in pancreatic β-cells
• miRNAs target a protein kinase mRNA resulting in its
14
silencing.
• miRNAs have been extensively studied in
Arabidopsis thaliana, a flowering plant that is a
member of the mustard family.

• In Arabidopsis, there are over 160 genes that

encode miRNAs. There are several mRNAs
known to be targeted for destruction by miRNAs.
– miRNA targets are the HD-ZIPIII class transcription
factors
– Some HD-ZIPIII class transcription factors instruct the
cells on the top surface of a leaf, while others instruct
the cells on the bottom of the leaf

15
 Cells located on the top and bottom surfaces of leaves
perform different functions

These cells specialize These cells specialize

in photosynthesis. in gas exchange.
miRNA destroys HD-ZIPIII factors involved in miRNA destroys HD-ZIPIII factors involved in
specializing cells for gas exchange. specializing cells for photosynthesis.

Some HD-ZIPIII class transcription factors instruct the cells on the top
surface of a leaf, while others instruct the cells on the bottom of the
leaf. This is because miRNAs regulate which HD-ZIPIII class
transcription factors are expressed on the top or bottom leaf surfaces. 16
miRNAs control gene expression by base pairing with specific mRNAs, essentially
affecting their stability & translation.

The precursor miRNA (70-90 nts) is

processed in the nucleus to form a
mature miRNA (24-26 nts).

miRNA then assembles with a set of

proteins into a complex called RISC.

The miRNA then guides the RISC to

mRNAs that have a complementary
nucleotide sequence.

Depending on how extensive the

region of complementarity is, the
target mRNA is either rapidly
degraded by a nuclease within the
RISC or transferred to an area of
the cytoplasm where other cellular
nucleases will destroy it.
17