Biology 340 Molecular Biology
Lectures 17 and 18
March 7 and 9, 2001
Regulation of Gene Expression in Prokaryotes
Reading: Chap. 10 pp. 341-358 Lodish et al. and
pp. 199-204 Weaver (1999) Molecular Biology. McGraw-Hill.
Outline:
1. Review of lactose (lac) operon
2. Cis regulatory elements
a. Promoter
b. Operator
3. Positive control of lac operon
4. Repressible operons: the trp operon
To understand the molecular bases for the fine adaptations of organisms
to their environment, it is essential to understand the way in which genes are
switched on and off. This aspect of molecular biology is known as gene
expression. We will especially focus on regulation of bacterial genes, because
the details are better worked out and because recombinant DNA technology
relies heavily on the use of bacterial regulatory systems.
Key elements of the lac operon:
1. Structural genes:
lac Z=-galactosidase gene
lac Y=permease gene
lac A=transacetylase gene
These are genes that occur in the order 5'--Z--Y--A--3' on the E. coli
chromosome and encode a polycistronic mRNA (multiple proteins are coded for
by a single mRNA).
2. Regulatory gene:
lac i=lactose repressor gene
--The lactose repressor is a protein that regulates the expression of the lac
operon according to the physiological state of the cell.
--In the presence of lactose, repressor binds to the inducer (a metabolized form
of lactose) and allows the lac structural genes to be expressed. OPERON ON
--When lactose is absent and glucose is abundant, the repressor binds to the
operator to shut down structural gene expression. OPERON OFF
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--The lac repressor is said to act in trans: the gene can be present anywhere in
the cell and need not be located on the same segment of DNA.
3. Regulatory elements:
P=promoter, site on DNA where RNA polymerase binds
O=operator, site on DNA where a regulatory protein (repressor) binds
These elements are said to act in cis: only on the adjacent segment of DNA.
Approaches used to study these elements:
Genetic:
--mutations in the promoter can alter the sequence recognized by RNA
polymerase; the operon cannot be transcribed.
--mutations in the operator (Oc mutants) alter binding by the repressor; thus the
operon no longer responds correctly to physiological signals.
Biochemical:
Methods used to track the interaction of regulatory proteins with DNA; useful for
studying or purifying prokaryotic or eukaryotic DNA binding proteins.
A. DNase I footprinting (See Fig. 10-6)
Method involves creating a DNA segment that is tagged on one end with
incubating the DNA in the presence or absence of a DNA binding protein,
followed by digestion of the DNA with DNase I. DNase I is unable to cleave DNA
if it is protected by bound protein. The protected region can be located by
electophoresis on a DNA sequencing gel.
32P,
B. Electrophoretic mobility shift assay (EMSA or more commonly, gel shift)
This assay uses an end labeled oligonucleotide containing the binding site
for the regulatory protein. Presence of the DNA binding protein can be detected
by a more slowly migrating band on a non-denaturing acrylamide gel (See Fig.
10-7). A "shifted band" consists of the protein bound to the oligonucleotide.
Biochemical analysis of the lac operon region (Fig. 10-8 and 10-9) identifies
binding sites for three regulatory protein in a short span of DNA. These include
RNA polymerase, lac repressor and another protein, CAP=catabolite activator
protein which also functions in lac regulation along with cAMP.
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Details of transcription initiation in E. coli
--Two regions in E. coli promoters are critical for binding RNA polymerase, called
-10 region and -35 region. Sequences in these regions bind to 70, the initiation
factor for RNA polymerase.
--The other subunits of RNA polymerase ( ') assemble on the sigma factor
(see Fig. 10-10).
--The DNA switches from a closed complex to an open complex as transcription
is initiated; after about 10 base pairs are copied, the 70 subunit is released, the
remaining subunits ( ') called the core polymerase, continue transcription
until a terminator is encountered.
--Promoter strength (the frequency with which transcription is initiated) depends
on the sequences at -10 and -35. The sequences of a number of strong E. coli
promoters are compared in Fig. 10-11 to give a consensus sequence. The lac
promoter is actually a weak promoter.
How does repressor work at the molecular level?
--repressor is a DNA binding protein
--repressor binds to the region between -5 and +20 on the DNA, which overlaps
the binding site of RNA polymerase on the DNA (-50 to +20).
--in vitro, when repressor is bound to DNA, it blocks RNA polymerase binding.
--addition of inducer to the repressor--DNA complex reduces the affinity of
repressor for the operator
--repressor is a dimer and binds an inverted repeat on DNA
3. Positive control of lac operon
E. coli can sense when glucose levels are dropping and it may be
necessary to break down other sugars. It does this through a positive control
mechanism involving cyclic AMP, a cyclic mononucleotide, and a regulatory
protein called catabolite activator protein (CAP).
See Fig. 10-16 for mechanism of positive control.
Summary:
A. When glucose is present, cAMP levels are low. The lac operon is turned off
by repressor.
B. When glucose is present and lactose is present, the lac operon is mostly off,
but some lac mRNA is made as needed to break down lactose.
C. When glucose is absent, cAMP levels are high. cAMP binds to CAP, which
changes its conformation and binds upstream in the promoter region (see Fig.
10-17), and enhances binding of RNA polymerase. The lac operon is fully on.
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4. Repressible operons: the tryptophan operon
E. coli biosynthetic operons, such as those for amino acids, are off when the
substance is available in the environment and yet are on to synthesize the
substance when it's needed.
Trp operon structural genes:
Trp EDCBA: genes for enzymes in tryptophan metabolism
Trp regulatory gene:
TrpR: repressor gene
Trp regulatory elements:
P=promoter
O=operator
Regulation of the trp operon is said to be repressible (see Fig. 7-26 of
Weaver)
--In the presence of little or no tryptophan, the operon is derepressed (turned on).
TrpR protein (repressor) is made, forms a dimer but is unable to bind DNA in the
absence of corepressor (tryptophan). Therefore RNA polymerase can bind to the
promoter and activate transcription.
--When tryptophan is at high levels, the operon is repressed (turned off).
Repressor is synthesized, dimerizes and binds to the corepressor (trp). The
repressor then binds to the operator and shuts down transcription.
Fine control of regulation by attenuation:
To allow fine adjustment of the levels of tryptophan in the cell, there is a second
mechanism that functions in control, called attenuation. Attenuation is a means
of prematurely terminating transcription of the Trp EDCBA genes. The more
tryptophan present, the greater the extent of attenuation, so fewer full length
transcripts are produced.
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