Biochemistry of Medicinals I
Phar 6151
Chapter SEVEN
Page 1
Biochemistry of Medicinals I Phar 6151 CHAPTER SEVEN
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
Regulation of Gene Expression
Regulation of gene expression in prokaryotes.
I.
•The rate
of protein synthesis in E. Coli varies 1000-fold in response to environmental changes
(temprerature, nutrients, etc.)
Gene activity is primarily regulated on the level of transcripttion
Prokaryotes regulate the expression of their genes in order to save the energy: instead of continuously
making unnecessary proteins, they are synthesiszed only when they are needed.
Two main types of prokaryotic genes:
1. Constitutively expressed genes (house-keeping genes) – always on
2. Regulated genes (inducible or repressible) –turned on and off
 -galactosidase of E. Coli is an example of inducible enzyme
E. coli can use lactose as carbon source by hydrolyzing lactose to galactose and glucose
catalyzed by  -galactosidase:
H H
H
-1,4-glucoside
bond

O O6
H H
H H
H
H
6
H

6
4
 -lactose
H
H O6
O H
H O
O
H
H
H
O
H
4
because this
O
H
O
O
1
1
O
O
4
O
H
OH is 
H
H
O
OH
OH
O
1 H
H
OH H
O
H
H
OH H 
OH
OH
 -D-Galactose unit
-D-Glucose unit
mammals)
Haworth projections 
-Galactosidase H2O
H
6
H O
O
H
H
H
O
1
+
OH
4
H
O OH
OH
O
H
H
O
H
H
O O6
H
4
H
OH
-D-Glucose unit -lactose (from the milk of
Chair structures 
for connection Gal(14)Glc-
H2O -Galactosidase
H
O6 H
OH
OH
H
-D-Galactose unit
H
H
H
O
H
O
6H
4
H
O
H HO
+ H O
O
H
1
OH H
H
H
1 OH
OH H 
When E. Coli is grown on glucose, it contains little  -galactosidase. In the presence of lactose, several E.
Coli enzymes are induced, including  -galactosidase, galactoside permease, and thio-galactoside transacetylase. Constitutive mutants synthesize large amounts of all three 3 proteins independent of the presence of
lactose.
Biochemistry of Medicinals I
Phar 6151
Chapter SEVEN
Page 2
Lac operon + Lactose : ◊ + ∆ = ◊∆  induce the Transcription
 -galactosidase,
galactoside permease, and thio-galactoside trans-acetylase are encoded by three
3 contiguous genes z, y, and a, respectively:
R
P O
z
y
a
STOP
• • •|••••|• • •|•••|•••|••••••••••|••••••••••••|••••••••••••|•••••••|• • • DNA anti-sence strand

◊∆
mRNA I gene◊∆ —— ——— ———
◊ repressor ∆  -Gal'ase

 -Gal-trans-Ac
LACTOSE
lac-permease

Splits LAC
into Gal+Glc
mRNA
Pumps LAC Not involved in
into the cell LAC metabolism
The expression of all three 3 genes is under control of a common element that contains control genes (P and
O) and a regulator gene (R).
Functions of various regions of the Lac operon ◊ :
P
Promoter
Binding site for RNA Pol
O
Operator
DNA control region that prevents transcription when repressor is bound
I
Regulator gene
Codes for the repressor protein ◊
z, y, and a Structural genes
Code for -galactosidase, galactoside permease, and
thio-galactoside trans-acetylase, respectively
The binding of repressor protein to operator prevents transcription of z, y, and a. In the presence of lactose,
allolactose is formed which serves as inducer. Another inducer is isopropylthiogalactoside (ITPG). Binding of
inducer to repressor protein prevents the latter from interaction with the operator. This leads to transcription of z,
y, and a and eventually the synthesis of the corresponding proteins.
I ——— P



O
i mRNA 
Z
O
4
H
O
H
H
H
H
O
H
H
4
1O

OH H O
H
Lac inducers:
1,6 Allolactose
O


repressor + inducer
H O H 6H
H
a
Y
H
H
O
O
H
1 OH
OH H 
O
Z
Y
repressor-IPTG
H 6H
H O H
6H
H
I — P
4
H
H

O
OH
H
deactivated
H
S
1
H
H
H H
H
OH
IPTG
a
Biochemistry of Medicinals I
Phar 6151
Chapter SEVEN
Interactions between lac repressor protein and operator sequence
·
·
·
·
Page 3
Lac repressor protein is a tetramer of identical 37 kD subunits
It tightly binds operator sequence (Kd = 10-13 M )
Finds operator site by diffusing along  DNA molecule
High selectivity for operator sequence (106)
Lac operator sequence has the same two-fold symmetry as the repressor protein.
The operator region directly interacting with the lac repressor is shown below:
5’…GAATTGTGACGCGGATAACAATTT…3’
3’…CTTAACACTGCGCCTATTGTTAAA…5’
Note the symmetrically related regions (highlighted)
Catabolite repression
·
·
·
·
The level of  -galactosidase is low  in the presence of glucose, the preferred E. Coli carbon source
Glucose suppresses lac operon transcription by lowering the concentration of cyclic AMP (cAMP)
The function of cAMP in bacteria is to activate CAP (catabolite gene activation protein)
cAMP-CAP complex binds promoter sites, stimulating the transcription of lac operon by bending
DNA and attracting RNA Polymerase to the promoter site
H N H
N
N
H
H
O
O P
O
N
O
H
O
N
H
O H
cAMP- cyclic Adenosine Mono-Phosphate
Conclusions
1. Lac is an inducible catabolic operon that is placed under dual control.
2. High  levels of expression require the presence of both lactose and cAMP
3. cAMP levels are lowered  in the presence of glucose, enabling E.Coli to conserve time and energy
needed for synthesis of lac proteins when they are not required