Download Lecture 10: Control of gene expression

Lecture 10: Control of gene expression
1. One gene –
one enzyme
hypothesis
2. Lac operon
3. Negative and
positive regulation
Lac repressor bound to DNA
The Nobel Prize in Physiology or Medicine 1958
"for their discovery that genes act by regulating definite
chemical events"
George Wells Beadle
1/4 of the prize
California Institute of
Technology (Caltech)
Edward Lawrie Tatum
1/4 of the prize
Rockefeller Institutute for
Medical Research, NY
"for his discoveries
concerning genetic
recombination and the
organization of the genetic
material of bacteria"
Joshua Lederberg
1/2 of the prize
University of Wisconsin
Madison, WI
Experiments of Beadle and Tatum, in 1940-ies, were the
first to shed light on general mechanisms of the gene
function.
They studied mutations in the arginine biosynthetic
pathway in Neurospora.
Arginine has two related substances:
citrulline and ornithine that are converted into arginine
by the pathway that must involve several enzymes:
Enz. X
Enz. Y
Enz. Z
Precursor -----> Ornithine ------> Citrulline -----> Arginine
Search for mutants
in amino acid biosynthesis
Growth of some arg- mutants can be supplemented
not only by arginine, but also by related compounds:
Mutant
arg 1
arg 2
arg 3
Ornithine
+
-
Citrulline
+
+
-
Arginine
+
+
+
(plus means growth, and minus means no growth)
The compound which is made the latest (Arg)
supports growth of the largest number of mutants
The compound which is made the earliest (Orn)
supports growth of smallest number of mutants
Enz. X
Enz. Y
Enz. Z
Precursor -----> Ornithine ------> Citrulline -----> Arginine
how to explain the observed growth pattern?
Mutant
arg 1
arg 2
arg 3
Ornithine
+
-
Citrulline
+
+
-
Arginine
+
+
+
- they hypothesized that each mutant is deficient in production of certain enzyme of the
pathway, e.g. arg1 could not produce Enzyme X. Then this mutant cannot convert the
precursor into ornithine. But given ornithine, it still can make citrulline and arginine.
Enz. X
Enz. Y
Enz. Z
Precursor -----> Ornithine ------> Citrulline -----> Arginine
arg1
Mutant
arg 1
arg 2
arg 3
arg2+
Ornithine
+
-
arg3+
Citrulline
+
+
-
Arginine
+
+
+
- mutant arg2 could not produce Enzyme Y. It can make ornithine (arg1+ allele) but cannot
convert it into citrulline. That’s why its growth is not supported by ornithine. Citrulline, however,
supports
Enz. X
Enz. Y
Enz. Z
Precursor -----> Ornithine ------> Citrulline -----> Arginine
arg1+
Mutant
arg 1
arg 2
arg 3
arg3+
arg2
Ornithine
+
-
Citrulline
+
+
-
Arginine
+
+
+
- mutant arg3 could not produce Enzyme Z. It can make ornithine and citrulline but cannot
convert citrulline into arginine. That’s why its growth is not supported by ornithine or citrulline
and is supported only by arginine.
Enz. X
Enz. Y
Enz. Z
Precursor -----> Ornithine ------> Citrulline -----> Arginine
arg1+
Mutant
arg 1
arg 2
arg 3
arg2+
Ornithine
+
-
arg3
Citrulline
+
+
-
Arginine
+
+
+
Enz. X
Enz. Y
Enz. Z
Precursor -----> Ornithine ------> Citrulline -----> Arginine
arg1+
arg2+
arg3+
‘One gene – one enzyme’ hypothesis:
genes control biosynthetic pathways by
encoding enzymes that catalyze specific
steps of the pathways
Growth of E. coli with two energy sources: glucose and lactose
Phase II: utilization of lactose
Phase I: utilization of glucose
... nor is the metabolic chart
Ability of E.coli cells to utilize lactose depends on
production of the enzyme β-galactosidase
Phenotype
Minimal Medium with glucose
Lac+(inducible)
Lac- (negative)
Lac++ (constitutive)
- (no enzyme)
+
Minimal Medium with lactose
+ (enzyme is produced)
+
lac operon
A group of genes that are regulated
together and are closely linked to
each other is called an operon
Genes of the same operon are
transcribed together as a
polycistronic (= polygenic) mRNA
Lac operon is located at 8 min of the
E. coli chromosome map. It contains
three genes lacZ, lacY and lacA. The
respective three enzymes of the lac
operon are induced simultaneously.
lacP (promoter) and lacO (operator)
are the operon’s regulatory regions
lacZ- or lacY- mutations cause Lac- phenotype
lac operon
An additional class of mutations was found
close to lac operon. These mutations caused
constitutive phenotype (Lac++), and it was
concluded that they affect ‘inducibility’ locus.
Hence the name lacI. The lacI gene
encodes lac repressor that inhibits the lac
lacZ- or lacY- mutations cause Lac- phenotype
operon. The operon is derepressed by
lacI- mutations case Lac++ phenotype
lactose in the medium.
The Nobel Prize in Physiology or Medicine 1965
"for their discoveries concerning genetic control of enzyme and
virus synthesis"
François Jacob
b. 1920
Jacques Monod
1910-1976
André Lwoff
1902-1994
lac repressor can reversibly attach to lac operon
Lac+ phenotype
lactose
absent
lactose
present
DNA –binding site
Normal lac-repressor can interact with (bind to):
1) DNA (lac-operon) and 2) inducer (lactose)
lactose –binding site
Mutant forms of lac repressor:
cannot attach to lac operon (lacI-) or cannot detach from it (lacIS)
Lac++ phenotype
lactose may be
absent or present
Lac- phenotype
lactose may be
absent or present
Mutant lacIS repressor is dominant to normal lacI+
Meroploid F+ cell
Lac- phenotype
F’ lacI+ / lacIS lacZ+ (lacIS is in cis to lacZ+)
F’ lacIS / lacI+ lacZ+ (lacIS is in trans to lacZ+)
The same
phenotype Lac-
Normal lacI+ repressor is dominant to mutant lacIMeroploid F+ cell
Lac+ phenotype
Normal lacI+ repressor is dominant to mutant lacIMeroploid F+ cell
Lac+ phenotype
F’ lacI+ / lacI- lacZ+ (lacI+ is in trans to lacZ+)
F’ lacI- / lacI+ lacZ+ (lacI+ is in cis to lacZ+)
The same
phenotype Lac+
Mutant lacOC operator in cis to lacZ is epistatic to all types of
lac repressor
Lac++ phenotype
F’ lacI+ / lacOc lacZ+
F’ lacIS / lacOc lacZ+
F’ lacI- / lacOc lacZ+
Any type or configuration of
lacI, provided that
lacOC is in cis to lacZ+ 
the same phenotype Lac++
Mutant lacOC operator in trans to lacZ has no effect
F’ lacOc / lacO+ lacZ+
Now the phenotype depends
on the type of the repressor
present: no epistatic effect of
lacOC on alleles of lacI
Carrot and whip on the molecular level
cyclic AMP, a regulator
of many biological
processes
the formulas are not for memorization
glucose
cAMP
glucose
Glucose is low 
cAMP
 transcription is activated