Download Readings Problems Background Week 8

MCB 142
MAJOR ADVANCES IN UNDERSTANDING EVOLUTION AND HEREDITY
FALL 2015
WEEK 8: OCTOBER 27 AND 29
OCTOBER 27: CONTROL OF GENE EXPRESSION
Enzyme induction—the E. coli lac system. Negative control and the trans-acting repressor.
OCTOBER 29: MESSENGER RNA
The problem of information transfer from DNA to the site of protein synthesis. Kinetic evidence for a
short-lived intermediate. Discovery of messenger RNA.
Readings to be Discussed on Tuesday October 27

William Hayes 1967. The Mechanism of Bacterial Sexuality 1967. Endeavor 26: 33-38.

Arthur B. Pardee, François Jacob and Jacques Monod 1959. The Genetic Control and Cytoplasmic
Expression of Inducibility in the Synthesis of β-galactosidase by E. coli. Journal of Molecular
Biology 1: 165-178.
Read the article.
Readings to be discussed Thursday October 29

Arthur Pardee 1958. Experiments on the transfer of information from DNA to enzymes.
Experimental Cell Research, Supplement 6, 142-151. Read pages 148-150 starting from "Must
DNA form an Intermediate Catalyst?"

Sydney Brenner, François Jacob and Matthew Meselson 1961. An unstable intermediate carrying
information from genes to ribosomes for protein synthesisis. Nature 190: 576-581.
Read the article.

Francis Crick 1988. "What Mad Pursuit", Basic Books, NY. pp 116-121.
Read pages 116-121.

François Jacob 1988. "The Statue Within", Basic Books, NY.
Read pages 309-317.

Matthew Meselson 2014. François and "X". Research in Microbiology 165: 313-315.
Read the article.
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Study Questions
1. Stable heterozygotes for the lac operon can be obtained in which one copy of the operon is on the
bacterial chromosome and the other copy is on a stably inherited transmissible plasmid called F-lac.
(The technology for constructing such stable partial heterozygotes had not been developed until after
the experiments reported in the "PaJaMo" paper of this week's assignment.) What would be the
phenotype, inducible or constitutive, of each of the following F-lac heterozygotes:
a)
i-z-y-/i-z+y+
b)
i+z-y-/i-z+y+
Explain your answer in terms of the model of lac operon control proposed in the PaJaMo paper.
+
-
2. Which mating and which figure of the PaJaMo paper show that i is dominant to i ? Explain.
3. Why, without inducer added, does the production of β-galactosidase shut off at about 2 hours, as
seen in Figure 4?
4. Pardee, Jacob and Monod on page 175 describe an "inducer" model that they reject. Instead, they
favor the "repressor" model described on page 176. In addition to being more complex, the
"inducer" model predicts a certain class of lac-minus mutations that were not known to exist (and do
not exist). What would be the nature of such mutants on the "inducer" model? What properties
would be expected of such mutations?
5.
What evidence do Brenner et al. present to show that RNA made after phage infection is found on
ribosomes made before phage infection? What evidence do they present to show that no ribosomes
are made after cells are infected by phage T2?
6.
What are models I, II and III of Brenner et al. for the synthesis of phage protein? What specific
experimental observations argue for or against each model?
note: BJM express surprise that the standard dialysis procedure for reassociating 30S and 50S
ribosomal subunits into 70S ribosomes fails to reassociate what they believe to be the 30S and 50S
particles in the “B band”. The reason for this unexpected result is that CsCl strips some of the
protein subunits from the 50S and 30S particles, giving what are called core particles. Unlike 30S
and 50S particles, core particles do not reassociate. It was fortunate that they do not, as the
reassociated 70S ribosomes thereby formed would have been of various intermediate densities
(heavy 30 with light 50; light 30 with heavy 50) making the density gradient analyses difficult to
interpret.
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Microbial Genetics & Control of Gene Expression -- Some Background
As we saw last week, Crick's 1958 discussion of how the structure of genes
determines the structure of proteins does not address the problem of what
controls the expression of genes -- what determines whether a particular gene
is or is not expressed. The first coherent picture of how gene expression is
controlled emerged from studies of the E. coli lac operon conducted during
the 1950s at the Institute Pasteur in Paris, led by Jacques Monod and
François Jacob. By 1959, it had been determined that:
François Jacob
Jacques Monod
(1910-1976)
(1920-2013)
-- The enzyme β-galactosidase, required for the utilization of the sugar
lactose, is present at only very low levels in wild-type E. coli growing on a
carbon source other than lactose. But if lactose (glucosyl-β-D-galactoside) or certain other β-Dgalactosides such as isopropyl-thio-β-D-galactoside (IPTG) or methyl-thio-β-D-galactoside (TMG)
are added, the enzyme is produced in large amounts, amounting to about 5 percent of total protein
synthesis in steady state. The enzyme is therefore said to be inducible.
Although IPTG and TMG are good inducers they, unlike lactose, are not metabolizable. By using
IPTG or TMG in induction experiments and a carbon/energy source such as glycerol or maltose (but
not glucose, see “catabolite repression” below) instead of lactose, one is able to study induction
phenomena free of complicating effects caused by variation in the availability or utilizability of the
carbon/energy source.
-- The utilization of lactose also requires lac-permease, a specific protein that transports lactose,
IPTG and TMG into the cell.
-- The genes that specify the structure of β-galactosidase and the permease, designated z and y,
respectively, map very close to each other and are expressed coordinately, being part of the lac
operon (see below).
Arthur Pardee
(1921-)
-- A third gene, designated i, governs the inducibility of β-galactosidase and permease. It
maps very close to z and y. In Arthur Pardee, Jacob and Monod (1959), a seminal
paper that has come to be known as PaJaMo, the gene order is tentatively given as y i z.
(The correct order is i z y but the misassignment has no consequences for the conclusions
+
of PaJaMo.) The gene in wild-type (inducible) E. coli is designated i , a designation
given simply because it is the form of the gene found in wild-type E. coli, before it was
+
known whether i is dominant or recessive with respect to i-. Cells mutant for the i gene,
designated i , produce β-galactosidase and permease constitutively, i.e., even in the
absence of lactose, IPTG, TMG, or other inducer.
As described in the reading by Hayes, the method of interrupted mating of Hfr
("male") with F- ("female") bacteria, developed by François Jacob and Elie Wollman
(1957, C.R. Acad. Sci., Paris, 244:1840-) allowed the production of transient partial
diploids called merozygotes. (Transient, because after an hour or so the injected DNA
is either integrated into the bacterial genome, replacing the homologous DNA segment
or else is lost, in either case leaving the recipient cell and its descendants haploid
Elie Wollman
Elie Wollman
(1917-)
(1917-2008)
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again.) After mixing Hfr cells with F- cells the former begin to inject their chromosomes into the F- cells
(via rolling-circle DNA replication), starting from a specific site on the circular E. coli chromosome.
The site and the direction of transfer depend on the particular Hfr strain. Under the conditions employed
in PaJaMo, i, z and y enter the F- cells starting at about 10 minutes after mixing the Hfr and F- bacteria.
Owing to heterogeneity in the time when the Hfr cells attach to the F- cells and begin to inject their
chromosomes, the number of F- cells that have received the izy gene cluster continues to increase
steeply for an additional 20-25 minutes. Injection of an entire chromosome takes about 100 minutes and
may be interrupted at any time by agitation in a blender. (For an excellent description of bacterial
conjugation in addition to historical account in the Hayes reading see:
http://www.ncbi.nlm.nih.gov/books/NBK21942/ )
Technical Notes:
i) Streptomycin prevents protein synthesis in the Hfr strep-sensitive cells but does not prevent them
from mating and transferring genes. Because the streptomycin resistance gene in the Hfr strains
used in PaJaMo enter the F- recipient only very late in the Hfr X F- matings, the F- cells never
become sensitive to streptomycin in these experiments.
ii) The phage T6 lyses T6-sensitive Hfr cells and therefore prevents them from mating.
By conducting matings with various combinations of i, z and y genes, and testing for the production of
β-galactosidase in the presence and absence of inducer as a function of time after initiating the mating,
Pardee, Jacob and Monod demonstrated that i+ is trans-acting and dominant to i-. Thus, an i- cell that is
otherwise wild-type synthesizes β-galactosidase and permease at a high rate while injection into such a
cell of the i+ gene by an Hfr causes β-galactosidase synthesis gradually to shut down, taking about two
hours for complete shutdown. The simplest interpretation of these findings, the interpretation favored
by PJ&M, is that the i+ gene makes a diffusible product and that the product, designated the lac
repressor, prevents the production of β-galactosidase and permease. On this picture, the inducer, for
example IPTG, prevents the repressor from acting (by binding to it, as was discovered later), rendering
the repressor unable to bind to its target site. This allows the z and y genes to produce their products.
Thus, in the lac system, the wild-type state of the z and y genes is "off" and turning them on (induction)
is not the result of an interaction of the genes with an activator, the model considered but not preferred
by PJM, but is instead is the result of inactivating the repressor. The control of z and y gene expression
is therefore negative. Only later the repressor binding site was identified and its mode of action was
determined.
Two fundamental questions are posed at the end of the paper: (i) What is the chemical nature of the lac
repressor--is it a primary gene product (an RNA) or a secondary gene product (a protein)? (ii) does the
repressor act at the level of the gene or at the level of the cytoplasmic machinery that synthesizes βgalactosidase and permease (and a third gene in the lac operon, acetylase, not then known.) Unless the
proteins are made directly on the DNA, there must be a gene product that carries information for protein
synthesis from the gene to the site of protein synthesis, the ribosomes—a messenger. (Oddly, the word
“messenger” as used in PaJaMo refers to the repressor, not to the yet to be discovered mRNA.)
Within a year, cis-acting mutations were found immediately adjacent to z that made the expression of z
and y independent of the state of the i gene. These mutations defined a site designated the operator,
symbolized by O. Further mapping revealed the order i O z y. Jacob and Monod termed the operator
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and the closely linked genes under the control of the i gene the lac "operon", the first to be characterized
of several similar groups of closely linked genes under the control of a common control site. (There is a
third gene in the lac operon, specifying a transacetylase and designated a. The map order is i O z y a
and all three genes, z, y, and a are under the control of the same operator.)
Walter Gilbert
(1932-)
Mark Ptashne
1940-
Early genetic evidence (including the existence of nonsense
suppressor mutations in the i gene) implied that the repressor is a
protein. This was directly demonstrated by the isolation of the lac
repressor by Walter Gilbert and Benno Müller-Hill in 1966 at
Harvard who in the following year demonstrated its binding to
DNA of the wild-type lac operon, as expected if repressor acts at
the level of the gene, and the prevention of such binding by the
Benno Müller-Hill
inducer IPTG. At the same time, Mark Ptashne, also at Harvard,
(1933-)
isolated the phage lambda repressor, showing it to be a protein
that binds to lambda DNA. Further studies of these and other systems supported the
general picture of bacterial control genes that specify repressors that bind at specific
sites on the DNA and thereby prevent expression of a group of adjacent genes by
preventing transcription by the downstream passage of RNA polymerase. In the lac
system, the inducer combines with the repressor preventing it from binding to the
operator, resulting in activation of the genes of the lac operon. This arrangement
insures that the enzymes needed for utilization of an exogenous substance, in this
case lactose, are produced only when the substance is available.
A simple variation on this mechanism allows cells to avoid the wastage of producing enzymes involved
in the endogenous biosynthesis of a substance when the substance, for example an amino acid, is
available exogenously. In that case, the interaction of the exogenous substance with the repressor
facilitates rather than prevents the binding of the repressor to its operator, thereby silencing the genes
under its control. An example is the operon that includes a cluster of genes for the biosynthesis of the
amino acid tryptophan. In both sorts of control, binding of the repressor to its operator silences genes in
the corresponding operon, the essential difference being whether repressor binding to the operator is
prevented or facilitated by the exogenous metabolite. In both cases, therefore, it is the silencing of genes
rather than their activation that is the common feature of the control circuit.
In contrast to negative control accomplished by binding of a repressor to an operator, as in the lac and
tryp systems, there are also bacterial systems with positive control. A particularly well-known system of
positive control in E. coli is, somewhat awkwardly, designated catabolite repression. It functions to
boost the expression of the lac operon and other carbon source utilization genes, like the maltose operon,
etc., when glucose is absent and when the other carbon source is present. When glucose, the
metabolically preferred carbon source, and another carbon source, such as lactose, are both present, the
cell utilizes both. But when only the non-glucose carbon source is present, the efficiency of its
utilization is increased. The absence of glucose in the PaJaMo experiments therefore enhanced the
difference in lac operon expression between i- and i+ cells.
====
Already in 1952, it was found that when a saturating concentration of inducer is added to a growing
culture of wild-type E. coli (i+z+), β-galactosidase production reaches its maximum rate within a very
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few minutes, much less than the bacterial generation time (Monod, Pappenheimer, Cohen-Bazire 1952
Biochim. Biophys. Acta 9: 648-660). Citing this observation in a 1955 review, Monod wrote that the
rate of synthesis per bacterium is "constant and maximal from the start". Although not mentioned in the
1959 PaJaMo paper, and apparently not yet realized by anyone at the Institut Pasteur or elsewhere, this
observation poses the question of how it could be that the maximum rate of β-galactosidase synthesis is
established so rapidly? Is the enzyme made directly on the gene, thereby achieving maximum rate as
soon as repression is relieved? Or does the gene make an intermediate information carrier? But in that
case, how could the maximum rate of synthesis be achieved so rapidly? If there is a carrier, the rate of
enzyme synthesis should not reach maximum rate until the carrier reaches steady state concentration.
But how could that happen so fast? There were two possibilities. Either there is no carrier and βgalactosidase is made directly on the DNA or else the carrier is very unstable, with a half life
comparable to the short time required to reach the maximum rate of enzyme synthesis, less than 2-3
minutes, much less than the ~30-minute doubling time of the bacteria.
Within a year, the short-lived carrier, mRNA, was discovered, as we will discuss on Thursday.
Messenger RNA - Some Background
Experiments done as early as 1952 showed that beta-galactosidase synthesis starts and reaches full rate
almost immediately after the addition of inducer and subsequent experiments showed the same almost
immediate attainment of full-rate synthesis upon introduction of the z+ gene of an Hfr into an F-, as
shown by Arthur Pardee in the assigned reading. Similarly early experiments also showed that synthesis
of the enzyme by an induced culture stops immediately when inducer is removed.
The finding in such experiments of full rate synthesis almost immediately after inducer addition or gene
entry and its prompt cessation upon removing inducer could be explained if (1) there is no intermediate
and instead the enzyme is made directly on the DNA or if (2) there is an unstable intermediate with a
short half-life so that its concentration reaches steady-state very quickly after the operon is activated by
addition of inducer or by introducing the operon into an i- recipient cell in an Hfr X F- mating. At the
time, however, no such unstable intermediate, presumably an RNA of size great enough to specify an
entire peptide chain, was known. It was known, however, that protein synthesis takes place on
ribosomes. Could ribosomal RNA carry the information for proteins synthesis? And is ribosoma RNA
unstable?
But a number of observations appeared to be at variance with the hypothesis that ribosomal RNA is the
information carrier for protein synthesis:
i)
The ribosomal RNA of E. coli and other bacteria was found to be of just two sizes (1.5 kb
and 2.9 kb) while the size of bacterial proteins covers a wide range.
ii)
The AT/GC ratio of bacterial DNA is uniform within any given species of bacteria but ranges
widely, from about 0.3 to 3, depending on the species. But the nucleotide composition of
ribosomal RNA is essentially the same in all bacteria.
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iii)
Density labeling experiments showed that all or nearly all of the ribosomal RNA is stable
(Davern and Meselson 1960 J. Mol. Biol. 2: 153-160).
Thus, the above observations meant that the “messenger” (as it was eventually named by Jacob, who
initially called it “X”) had to be unstable and that it was not ribosomal RNA. The only other cellular
RNA known at the time was "soluble RNA" (transfer RNA), too small to code for most proteins. Then
what RNA was the short-lived messenger?
An important but initially unrecognized clue was the discovery in 1956 by Elliot Volkin and Lazarus
Astrachan at Oak Ridge National Laboratory in Tennessee that a short-lived RNA made in E. coli after
infection by phage T2, previously detected by Alfred Hershey, has a nucleotide composition
resembling the phage DNA (high AT, low GC) -- quite unlike that of its E. coli bacterial host, for which
AT equals GC (Virology 2: 149-161, 1956; Biochim Biophys Acta 29: 536-544, 1958.) At the time,
Volkin and Astrachan thought the labile RNA might be an intermediate for phage DNA synthesis
although, largely unnoticed, they did mention in a later paper (Volkin, Astrachan and Countryman
1958 Virology 6: 545-555), among other possibilities, that the newly synthesized RNA might be the
template for phage protein.
It was then, at a party in Sydney Brenner’s apartment in Cambridge on April 16, 1960 (Good Friday),
where Jacob recounted recent additional evidence for a short-lived messenger (including that in the
assigned Pardee reading) that Brenner and Crick suddenly realized the significance of the short-lived
RNA described by Volkin and Astrachan four years before.
A critical experiment to demonstrate the existence of a short-lived RNA that carries the information for
amino acid sequence to pre-existing ribosomes -- messenger RNA was then conceived by Brenner and
Jacob. E. coli would be grown several generations in heavy isotopic medium, making essentially all the
ribosomes heavy. It was already known that essentially all protein synthesized after phage infection is
phage protein and therefore that whatever template specified it was not present before infection. Upon
switching the cells to light isotopic medium and infecting with phage in the presence of radioactive
phosphorus (or radioactive uracil) and also with radioactive sulfur, it could then be asked if new RNA
and new protein appeared on old (heavy) ribosomes or on newly synthesized (light) ribosomes, if any.
If only on old ribosomes, this would rule out the possibility that the phage proteins are specified on new
ribosomes. A separate experiment, not using density labels, could be done to see if RNA made
subsequent to phage infection is found on ribosomes and if it resides there only briefly. The two sorts of
experiment together showed that there is a
species of RNA that appears only briefly
on the pre-existing ribosomes where new
protein is being made—and, further, that
no ribosomes are even made after phage
infection.
The obvious way to conduct such an
experiment was to employ equilibrium
density-gradient centrifugation with
density labels sufficiently different to
separate labeled and labeled ribosomes, a
Left to Right: Charles Steinberg, François Jacob, Max Delbruck,
Matthew Meselson, Ronald Rolfe, Gunther Stent, Sydney Brenner.
June 1960.
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separation that required the use of both heavy nitrogen (15N) and heavy carbon (13C). As 15N 13C yeast
extract made for growing E. coli had already been employed at CalTech in showing that ribosomal RNA
is stable (Davern and Meselson, 1960) and attempts to examine bacterial ribosomes by equilibrium
density gradient analtsis in CsCl solutions were already underway, Brenner and Jacob went to CalTech
to conduct the experiment together with Meselson. The outcome would validate the prediction of
Pardee (1958) and also of Jacob (1958), of a short-lived intermediate and show that it is a kind of RNA
distinct from ribosomal RNA and transfer RNA (Brenner, Jacob and Meselson, 1961). An authentic
account of the moment of realization in Brenner’s Cambridge apartment and the cliff-hanging
experiment in California that followed is given by Jacob in his autobiographic "The Statue Within".
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