Etty Widayanti, SSi. MBiotech.

Bagian Anatomi Sub Bagian Biologi

Fak. Kedokteran Univ. YARSI


Nov 2009
Regulation of gene expression
1. Not all genes are turned on (expressed)
all the time.
2. Control of the rate of protein or RNA
synthesis as an adaptive response to
stimuli.
In general, they are turned on
only when needed
Why regulate gene expression?
Regulation allows cells to respond
to environmental conditions by
synthesizing selected gene products
only when they are needed.
In Bacteria :
often involves nutrient utilization pathway

In Eukaryotes:
may involve the generation of specific proteins in
specific types of cells
The First Model: Lactose utilization in E. coli:

B-galactosidase
lactose galactose + glucose
(a disaccharide) H20 (energy)

Cells should make the needed proteins at the right time
Do not waste energy

cell does not waste energy making
enzymes it does not need.
Gene expression synthesis of a gene
product
1. Constitutive

2. Regulated / inductive
1. Constitutive gene expression
e.g. "housekeeping genes" like primase
ssDNA binding proteins
expression of genes at about the
same level under all environmental
conditions
2. Regulated gene expression
Control of the rate of protein or RNA
synthesis as an adaptive response to
stimuli.
induction: increase in gene expression
repression: decrease in gene expression
Adaptation and Induction

The presence of substrate, lactose, caused the appearance of
enzyme, B-galactosidase. Was this induction an "adaptation" of
the enzyme to substrate just as the bacterium "adapts" to
environment?
1. In absence of lactose 1-2 molecues/cell
In presence of lactose 100,000 molecules/cell

2. Synthesized nearly simultaneously and only after
lac mRNA becomes detectable.

3. Lactose (and analogs) is inducer
B-galactosidase (and permease) are inducible

How does a cell "KNOW" what to make?
Monod, Jacob, Lwoff sought to explain induction
GENETICALLY
Control of Gene Expression in Bacteria
1. The lac operon (genetics)

2. Promoters and repressors

3. Other operons
1. Structural genes: lac Z, Y, A (transport & metabolism)
Regulatory elements: the lac I gene- repressor
the lac O operator
the lac P promoter

2. lac Z, Y, A in a single mRNA polycistronic

3, Promoter is adjacent to operator (lac P
-
- no mRNA)

4. lac I protein binds to operator represses transcription

5. Inducers, e.g. Lactose, bind to and inactivate repressor
The Operon Model
Lac Operon
Example of gene induction:
Regulation of the lac operon
The lac operon is a group of genes used for
catabolism of the sugar lactose
Z Y A
lac genes
promoter
operator
The lac operon of E. coli
When lactose is available, E. coli
induces expression of lac operon.
When lactose is unavailable,
the catabolic enzymes are
NOT needed.
The lac operon is expressed
at only very low levels.
lac repressor is allosteric: it has two
different conformations:

1. In presence of inducer, it does not bind DNA

2. In absence of inducer, it binds strongly to lac
operator DNA
Negative Control of the lac Operon by the
lacI Repressor
Glucose indirectly inhibits lac expression
glucose lac
if glucose is high cAMP is low
Positive Control of the lac Operon:
CAP and Catabolite Repression
Lac Z
Remember lactose galactose + glucose
If lactose & glucose are present - no lacZ is made until
glucose is depleted.
How?

High cAMP is necessary for activation of lac operon
cAMP is bound by CAP (catabolite activator protein)
cAMP-CAP binds to distal part of promoter and facilitates
transcription
Glucose is normal energy and carbon source.

Cell has "back-up" system to use lactose (lac).

Even if lactose is present, it won't bother to make
lacZ if glucose is present.

Even if no glucose is present, operon isn't unduced
until lactose appears.

Lactose inhibits inhibition of lac expression
The "Logic" of Operon Control and
Resource Utilization
General Theme:
A metabolite controls the expression of a battery of
genes that have evolved to utilize it (CATABOLISM)

Other Examples:
trp - tryptophan biosynthesis
ara - arabinose utilization
his - histidine biosynthesis
Operons
Think About:
Genetic logic of negative or positive control
i. e. repression & activation
1. trpE is first gene in operon
2. trpE mRNA has long leader (untranslated 5' region)
3. region of mRNA works as attenuator:
in presence of tryptophan, transcription is halted
about 140 bases into mRNA
in absence of tryptophan, transcription continues
Tryptophan Operon
Mechanism is complex, but logical
leader sequence contains short 11 amino acid peptide with
two trp residues
when trp is abundant, trp-tRNA can be used to translate
leader mRNA which terminates transcription
when trp is limiting, translation stalls and transcription is
permitted
The trp operon is regulated at two levels
P
O
E D C B A
1. repression by trp repressor (on/off)
genes encoding the enzymes used
for tryptophan biosynthesis
2. attenuation (fine tuning by transcriptional
termination)
R
R
Gen trp R Aporepressor
Trp = co repressor
When the level of trp is high, trp
does not have to be synthesized.

Trp binds the repressor, and the
repressor binds DNA and prevents
RNA pol binding to the promoter.
Trp Attenuation
The latest estimates are that a human
cell, a eukaryotic cell, contains
approximately 35,000 genes.

How is gene expression regulated?
There are several methods
used by eukaryotes
Transcription Control
The most common type of genetic regulation
Turning on and off of mRNA formation

Post-Transcriptional Control
Regulation of the processing of a pre-mRNA into a mature
mRNA

Translational Control
Regulation of the rate of Initiation

Post-Tranlational Control
Regulation of the modification of an immature or inactive
protein to form an active protein

DNA
new RNA transcript
mRNA
mRNA
polypeptide chain
active protein
A. Transcription
B. mRNA processing
C. mRNA transport
D. translation
E. Protein processing
nucleus
cytoplasm
Binding of transcription factors to
special sequences in DNA slows or
speeds transcription.

Chemical modifications and chromosome
duplications affect RNA polymerases
physical access to genes.
A.Transcription
New mRNA cannot leave the nucleus
before being modified, so controls over
mRNA processing affect the timing of
transcription.

Controls over alternative splicing
influence the final form of the protein.
B. mRNA processing
RNA cannot pass through a nuclear pore
unless bound to certain proteins.

Transport protein binding affects where
the transcript will be delivered in the
cell.
C. mRNA transport
An mRNAs stability influences how long it is
translated.

Proteins that attach to ribosomes or initiation
factors can inhibit translation.

Double-stranded RNA triggers degradation of
complementary mRNA.
D. Translation
A new protein molecule may become
activated or disabled by enzyme-
mediated modifications, such as
phosphorylation or cleavage.

Controls over these enzymes influence
may other cell activities.
E. Protein processing
Transcriptional Control
RNA polymerase II (pol II) is a complex of some
10 different proteins.
The start site is where transcription of the gene
into mRNA begins.
Transcription start site
Transcriptional Control

The basal promoter contains a sequence of 7 bases (TATAAAA) called
the TATA box (this is very similar to the -10 box or Pribnow box found
in prokaryotes) .

It can be bound by Transcription Factor IID (TFIID read TF2D)
which is a complex of some 10 different proteins including
- TATA-binding protein (TBP), which recognizes and binds to the
TATA box
- other protein factors which bind to TBP - and each other - but not
to the DNA.

The basal or core promoter is found in all protein-encoding genes. This
is in sharp contrast to the upstream promoter whose structure and
associated binding factors differ from gene to gene (i.e. they are unique
to each specific gene).
The basal promoter
PROMOTER PROKARIOT & EUKARIOT
Just how do proteins bind to DNA?
DNA : Protein and Protein : Protein interactions are
important for transcription factor function.

Note modular structure of transcription factors:
one part of the protein is responsible for DNA
binding, another for dimer formation, another for
transcriptional activation (i.e. interaction with basal
transcription machinery).
Dimer formation adds an extra element of complexity and
versatility. Mixing and matching of proteins into different
heterodimers and homodimers means that three distinct
complexes can be formed from two proteins.

Diverse in nature, but several common structures are
found:
- Helix-turn-helix (homeodomain) - three different
planes of the helix are established and bind to the
grooves of the DNA
- Zinc fingers - cystine and histidine residues bind to
a Zn2+ ion, looping the amion acid into a finger-like
chain that will rest in the grooves of DNA
- Leucine zipper - dimers result from leucine residues
at every other turn of the a-helix. When the a-
helical regions form a leucine zipper, the regions
beyond the zipper form a Y-shaped region that grips
the DNA in a scissors-like configuration

REFERENCES
Campbell, N.A., Reece, J.B. and Mitchell, L.G. 2004.
Biologi. Jilid ke-3. Ed ke-5. Penerbit Erlangga,
Jakarta.
Lehninger, A.L. 1982. Dasar-dasar biokimia. Jilid 3.
Terj dari Principles of biochemistry, oleh
Thenawidjaya, M. Penerbit Erlangga, Jakarta.
Starr, C., Taggart, R., Evers, C. and Starr, L. 2009.
Cell biology and genetics. Biology: The unity and
diversity of life. 12th ed. Brooks/Cole, Belmont.
Yuwono, T. 2005. Biologi molekular. Penerbit Erlangga,
Jakarta.