Download Gene Regulation in Prokaryotic Cells

Bio300
Handout #15
Fall, 2004
Gene Regulation in Prokaryotic Cells
(Chapter 10)
I. The basics of prokaryotic transcriptional regulation
A. Key Words: promoter, operator, activator, repressor. The binding of regulatory
proteins can either activate or block transcription – Fig. 10-2.
B. Domains of DNA binding proteins (activators or repressors)
• DNA binding domain
• Allosteric site and allosteric effectors
allosteric: of, relating to, undergoing, or being a change in the shape and
activity of a protein (as an enzyme) that results from combination with
another substance at a point other than the chemically active site.
(Wester’s Dictionary)
allosteric effectors : small molecules interacting with a allosteric site.
• Some activator or repressor proteins must bind to their allosteric
effectors before they can bind DNA. Others are opposite – loosing the
ability to bind DNA once they bind effectors – Fig. 10-3.
II. An overview of the lac regulatory circuit in E. coli
A. Operon (one promoter controlling several genes) – Fig. 10-4
C. Structural genes (lacZ, lacY and lacA) vs. the regulatory gene (lacI).
D. Two DNA docking sites – promoter and operator
B. The induction of the lac system.
E. The operon is off normally – binding of the lactose-free Lac repressor to
the operator sequence.
F. Binding of lactose of its analogs to the repressor protein results in an
allosteric transition so that the repressor cannot bind the operator DNA
anymore.
G. A RNA polymerase binds to the promoter and turns on transcription.
III. Discovery of the lac system of negative control
A. Jacob and Monod found that genes were controlled together.
H. Genetic evidence for the presence of gene I, which regulates the expression
of gene lacZ encoding β-galactosidase. β-Galactosidase is an inducible
enzyme. New enzymes (containing radioactively labeled amino acids) were
induced as early as 3 min after the inducer was added.
I. Several mutants that couldn’t be complemented by one another – different
genes.
J. The lacZ, lacY and lacA genes were very closely linked on the chromosome,
as shown by recombination mapping.
B. Genetic evidence for the operator and repressor
K. Used the non-metabolizable induce, isopropyl-β-D-thiogalactoside (IPTG) –
Fig. 10-7.
L. Assessment of dominance. Created partial diploids by using F’ factors.
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Bio300
Handout #15
Fall, 2004
M. Cis-acting Oc constitutive mutants – Table 10-1. The wild type lacY gene is
cis to the wild type operator and the wild type lacZ gene is cis to the Oc
constitutive mutant – Strain 4.
N. This cis-acting property of O suggests that it acts simply as a protein-binding
site (a piece of DNA) and makes no gene product – Fig. 10-8.
O. I+ is dominant to I-; I+ is trans-acting. The I+ gene product can regulate all
structural lac operon genes, whether in cis or trans (residing on different
DNA molecules) because the protein product of the I gene is able to diffuse
and act on both operators in the partial diploid – Table 10-2, Fig. 10-9.
C. Genetic evidence for allostery
P. Suppressor (Is) mutations. Is repressors cannot bind the IPTG inducer, but are
still able to bind to the operator – Table 10-3, Fig. 10-10.
Q. Is mutation are dominate to I+. So the repression is constitutive; cannot be derepressed by IPTG.
VI. Catabolite repression of the lac operon: positive control
A. Choosing the best sugar to metabolize
R. A breakdown product, or catabolite, of glucose prevents activation of the lac
operon by lactose.
S. In the presence of the hunger signal, cyclic AMP (cAMP), CAP (catabolite
activator protein) protein (activator) binds to the promote to enhance the
transcription of the lacZ gene (and others on the operon) – Fig. 10-13 b
T. cAMP-CAP and RNA polymerase mutually stimulate the binding of
themselves to the lac control region via cooperative binding.
4. The activity of adenylate cyclase (which catabolizes the production of cAMP
from ATP) is down-regulated by glucose – Fig. 10-13a.
B. The structures of target DNA sites
U. The CAP-cAMP binding to a site different from that for the lacI repressor,
although both bind to the 5’ end of the operon – Fig. 10-14.
V. The bending of DNA, which is promoted by the CAP-cAMP complex, may
aid the binding of RNA polymerase to the promoter.
W. CAP and DNA polymerase bind directly adjacent to each other on the lac
promoter – Fig. 10-16.
X. A summary of the lac operon – Fig. 10-17.
D. Comparison of repression and activation – Fig. 10-18.
V. Dual positive and negative control: the arabinose operon.
A. The structure of the ara operon – Fig.10-19
B. Regulation:
• In the presence of arabinose, the AraC protein binds to the araI region (the
initiator region, containing both an operator site and a promoter) and the
CAP-cAMP complex binds to a site adjacent to araI, leading to the
expression of the araB, araA and araD genes (three structural genes that
encode the metabolic enzymes to break down the sugar arabinose).
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Bio300
Handout #15
•
Fall, 2004
In the absence of arabinose, the AraC protein functions as a repressor by
binding to both the araI and araO regions, forming a DNA loop. This
binding prevents transcription of the ara operon. Fig. 10-20.
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