Download 14.1 The lacI Gene Encodes a Diffusible Repressor

Robert J. Brooker - Genetica
Esperimento di genetica 14.1
The lacI Gene Encodes a Diffusible
Repressor Protein
Now that we have an understanding of the lac operon, let’s consider
one of the experimental approaches that was used to elucidate its
regulation. In the 1950s, Jacob, Monod, and their colleague Arthur
Pardee had identified a few rare mutant strains of bacteria that had
abnormal lactose adaptation. One type of mutant, designated lacI–,
resulted in the constitutive expression of the lac operon even in the
absence of lactose. From this observation, the researchers incorrectly hypothesized that the lacI– mutation resulted in the synthesis
of an internal inducer, making it unnecessary for cells to be exposed
to lactose for induction (Figure EG14.1.1). By comparison, Figure
EG14.1.1b shows the correct explanation. A loss-of-function mutation in the lacI gene prevented the lac repressor protein from inhibiting transcription. At the time of their work, however, the function
of the lac repressor was not yet known.
To further explore the nature of this mutation, Jacob, Monod,
and Pardee applied a genetic approach. In order to understand their
approach, let’s briefly consider the process of bacterial conjugation
(described in Chapter 6). The earliest studies of Jacob, Monod, and
Pardee in 1959 involved matings between recipient cells, termed F–,
and donor cells, which were Hfr strains that transferred a portion of
the bacterial chromosome. Later experiments in 1961 involved the
transfer of circular segments of DNA known as F factors. We will
consider the latter type of experiment here. Sometimes an F factor
also carries genes that were originally found within the bacterial
chromosome. These types of F factors are called F' factors (F prime
factors). In their studies, Jacob, Monod, and Pardee identified F'
factors that carried the lacI gene and portions of the lac operon.
These F' factors can be transferred from one cell to another by bacterial conjugation. A strain of bacteria containing F' factor genes is
called a merozygote, or partial diploid.
The production of merozygotes was instrumental in allowing
Jacob, Monod, and Pardee to elucidate the function of the lacI gene.
This experimental approach has two key points. First, the two lacI
genes in a merozygote may be different alleles. For example, the
lacI gene on the chromosome may be a lacI– allele that causes constitutive expression, while the lacI gene on the F' factor may be
normal. Second, the genes on the F' factor and the genes on the bacterial chromosome are not physically adjacent to each other. As we
now know, the expression of the lacI gene on an F' factor should
produce repressor proteins that could diffuse within the cell and
eventually bind to the operator site of the lac operon located on the
chromosome.
Figure EG14.1.2 shows one experiment of Jacob, Monod, and Pardee in which they analyzed a lacI– mutant strain that was already
known to constitutively express the lac operon and compared it to
the corresponding merozygote. The merozygote had a lacI– mutant
gene on the chromosome and a normal lacI gene on an F' factor.
These two strains were grown and then divided into two tubes each.
In half of the tubes, lactose was omitted. In the other tubes, the
strains were incubated with lactose to determine if lactose was
needed to induce the expression of the operon. The cells were lysed
by sonication, and then a lactose analogue, β-ONPG, was added.
This molecule is colorless, but β-galactosidase cleaves it into a
product that has a yellow color. Therefore, the amount of yellow
color produced in a given amount of time is a measure of the
amount of β-galactosidase that is being expressed from the lac operon.
THE HYPOTHESIS
The lacI– mutation results in the synthesis of an internal inducer.
FIGURE EG14.1.1 Alternative ideas to explain how a lacI– mutation could cause the constitutive expression of the lac operon. (a) The hypothesis of
Jacob, Monod, and Pardee. In this case, the lacI– mutation would result in the synthesis of an internal inducer that turns on the lac operon. (b) The correct
explanation in which the lacI– mutation eliminates the function of the lac repressor protein, which prevents it from repressing the lac operon.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
TESTING THE HYPOTHESIS — FIGURE EG14.1.2 Evidence that the lacI gene encodes a diffusible repressor protein.
Starting material: The genotype of the mutant strain was lacI– lacZ+ lacY+ lacA+. The merozygote strain had an F' factor that was lacI+
lacZ+ lacY+ lacA+, which had been introduced into the mutant strain via conjugation.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
THE DATA
Strain
Mutant
Mutant
Merozygote
Merozygote
Addition of Lactose
No
Yes
No
Yes
Amount of β-Galactosidase
(percentage of parent strain)
100%
100%
<1%
220%
INTERPRETING THE DATA
As seen in the data, the yellow production in the original mutant
strain was the same in the presence or absence of lactose. This result is expected because the expression of β-galactosidase in the
lacI– mutant strain was already known to be constitutive. In other
words, the presence of lactose was not needed to induce the operon
due to a defective lacI gene. In the merozygote strain, however, a
different result was obtained. In the absence of lactose, the lac operons were repressed—even the operon on the bacterial chromosome. How do we explain these results? Because the normal lacI
gene on the F' factor was not physically located next to the chromosomal lac operon, this result is consistent with the idea that the lacI
gene codes for a repressor protein that can diffuse throughout the
cell and bind to any lac operon. The hypothesis that the lacI– mutation resulted in the synthesis of an internal inducer was rejected. If
that hypothesis had been correct, the merozygote strain would have
still made an internal inducer, and the lac operons in the merozygote would have been expressed in the absence of lactose. This result was not obtained.
The interactions between regulatory proteins and DNA sequences illustrated in this experiment have led to the definition of
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
two genetic terms. A trans-effect is a form of genetic regulation
that can occur even though two DNA segments are not physically
adjacent. The action of the lac repressor on the lac operon is a
trans-effect. A regulatory protein, such as the lac repressor, is called
a trans-acting factor. In contrast, a cis-acting element is a DNA
segment that must be adjacent to the gene(s) that it regulates, and it
is said to have a cis-effect on gene expression. The lac operator site
is an example of a cis-acting element. A trans-effect is mediated by
genes that encode regulatory proteins, whereas a cis-effect is mediated by DNA sequences that are bound by regulatory proteins.
Jacob and Monod also isolated constitutive mutants that affected the operator site, lacO. Table EG14.1.1 summarizes the effects of mutations based on their locations in the lacI regulatory
gene versus lacO and their analysis in merozygotes. As seen here, a
loss-of-function mutation in a gene encoding a repressor protein has
the same effect as a mutation in an operator site that cannot bind a
repressor protein. In both cases, the genes of the lac operon are constitutively expressed. In a merozygote, however, the results are
quite different. When a normal lacI gene and a normal lac operon
are introduced into a cell harboring a defective lacI gene, the normal lacI gene can regulate both operons. In contrast, when a lac
operon with a normal operator site is introduced into a cell with a
defective operator site, the operon with the defective operator site
continues to be expressed without lactose present. Overall, a mutation in a trans-acting factor can be complemented by the introduction of a second gene with normal function. However, a mutation in
a cis-acting element is not affected by the introduction of another
cis-acting element with normal function into the cell.
TABLE EG14.1.1 A Comparison of Loss-of-Function Mutations in the
lacI Gene versus the Operator Site
Chromosome
Wild type
lacI–
lacO–
lacI–
lacO–
F’ factor
None
None
None
lacI+ and a
normal lac
operon
lacI+ and a
normal lac
operon
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Expression of the lac Operon
With Lactose Without Lactose
100%
<1%
100%
100%
100%
100%
200%
<1%
200%
100%