Download Predicted Existence of Messenger RNA: The Operon Model Until

Predicted Existence of Messenger RNA: The Operon Model
Until 1960 it was thought that rRNA represented the set of templates for
protein synthesis. Jacob and Monod questioned this idea because rRNAs
have homogenous sizes (5S, 16S, and 23S in bacteria) whereas proteins are
of heterogeneous size. From the analysis of mutant bacteria which were
altered in their ability to control lactose metabolism, they correctly predicted
the existence of mRNA.
Bacterial Lactose Metabolism:
Lactose use is controlled in bacteria by three enzymes whose genes are
adjacent on the chromosome (operon), one of these proteins is βgalactosidase which hydrolyzes lactose and other β-galactosides.
- When grown on glucose as a energy source- lactose enzymes are
very low in bacteria.
- When shifted to lactose rich media- these enzymes are highly
expressed. Removal of lactose rapidly leads to very low expression of
the proteins.
These rapid changes suggested that the template for producing these
enzymes was rapidly synthesized on demand, and as rapidly degraded when
a continued stimulus (lactose) was absent. Because rRNAs are stable, they
reasoned that they were not the intermediates in this information transfer.
They used a variety of bacterial mutants: Some expressed the enzymes
at high levels in the absence of lactose, others never made these enzymes in
the presence of lactose. These results led to the idea of a molecular
REPRESSOR that regulated gene expression by binding to a specific
OPERATOR sequence on DNA to block RNA synthesis.
Based on these studies, they formulated a unifying hypothesis:
They proposed that transcription involved copying a DNA strand to give an
RNA of complementary sequence, and that this process was controlled at the
initiation stage. Hypothetical regulatory elements called repressors and
operators controlled the synthesis of other hypothetical RNAs termed
messenger RNA.
Regulation of Prokaryotic Transcription:
If all cells have the same DNA content, and the DNA of a cell specifies its
activities (what enzymes it makes) and characteristics (what effect these
enzymes have), why aren't all cells the same?
We know that there are different cell types in our bodies, and that the
activities of these cells changes with time. How do these cells know which
gene products are needed and when they are needed or not needed?
This question as it applied to large, complex organisms like humans was very
daunting for scientists in the first half of the 20th century. Francois Jacob and
Jacques Monod approached the problem from a more basic and simple
perspective. How did single-celled prokaryotes like E. coli know how to
respond to their environments?
Each environmental cue generates a specific response, with specific
proteins and reactions. For example, a bacterium can use several different
sources of nitrogen. Some bacteria can incorporate diatomic nitrogen gas
from the air, or incorporate ammonia from their surroundings, or break the
amine group from the end of an amino acid like glutamine. It is much easier
and less energy costly for the cell to use the nitrogen from glutamine than to
fix nitrogen gas from the air. These two processes require very different
enzymes to allow them to occur. If there is glutamine around, the cell should
be able to shut off the enzymes that are involved in the incorporation of
nitrogen gas. In fact, it shouldn't have to waste the energy to synthesize
these enzymes at all (remember how much energy is needed to carry out
translation). How can the cell "turn off" the synthesis of proteins from its DNA,
when the moment calls for it?
How are RNA levels regulated?
Primarily regulated by controlling how frequently each gene is transcribed to
RNA.
Initiation of Transcription is the major control point:
So the central question is:
What determines the frequency at which a specific gene is
transcribed?
The Transcription rate is controlled by proteins that bind to specific DNA
sequences in the promoters of a particular gene.
Promoter sequences are usually found at the 5' end of a gene relative to the
coding region.
These proteins are called "Transcription Factors". Each recognizes a
particular DNA sequence.
Binding of a transcription factor to a promoter sequence determines whether
or not RNA polymerase binds to and initiates transcription of a particular
gene.
Methods for regulating gene expression:
The cell could somehow selectively inhibit transcription of the gene. The
mRNA for this gene would never be made.
The cell could selectively degrade the mRNA as soon as it was made,
preventing it from being translated into protein.
The cell could selectively prevent translation on an otherwise stable mRNA.
The cell could selectively degrade the translated protein so that it couldn't
waste energy trying to catalyze reactions that the cell has no need for at that
time.
In all these cases, the cell has to have some way of shutting off the
unwanted protein selectively and leaving on the other genes in the cell. As
you can imagine, in terms of energy cost, it is better to shut off the process
as early as possible, so that no energy is wasted in mRNA and protein
synthesis.
This type of early-intervention control is called transcriptional regulation,
since expression of the gene is regulated at the level of mRNA synthesis, or
transcription.
Jacob and Monod were the first scientists to elucidate a
transcriptionally regulated system. They worked on the lactose
metabolism system in E. Coli. When the bacterium is in an environment
that contains lactose:
It should turn on the enzymes that are required for lactose degradation.
These enzymes are:
beta-galactosidase:
This enzyme hydrolyzes the bond between the two sugars, glucose and
galactose. It is coded for by the gene LacZ.
Lactose Permease:
This enzyme spans the cell membrane and brings lactose into the cell
from the outside environment. The membrane is otherwise essentially
impermeable to lactose. It is coded for by the gene LacY.
Thiogalactoside
transacetylase:
This enzyme catalyzes the
acetylation of beta galactosides
that cannot be hydrolyzed by
beta galactosidase. Once
modified these molecules can
diffuse through the plasma
membrane and are eliminated
from the cell. LacA
These three enzymes appear
adjacent to each other on the E.
Coli genome. They are preceded
by a region that is responsible for
the regulation of the lactose
metabolic genes. These genes
are thus part of an Operon that
is transcribed from a single
promoter to produce a large
mRNA molecule containing three
separate protein-coding regions.
It would seem that the cell would
want to turn these genes on
when there is lactose around and
off when lactose is absent. But
we will see that regulation is
more complicated than that (even
for a very simple system in a
"simple" organism).
A bacterium's prime source of
food is glucose, since it does not
have to be modified to enter the
respiratory pathway. So if both
glucose and lactose are available, the bacterium wants to turn off lactose
metabolism in favor of glucose metabolism. There are sites upstream of the
Lac genes that respond to glucose concentration.
This assortment of genes and their regulatory regions is called the Lac
operon.
Element
Operator (LacO)
Promoter (LacP)
Repressor (LacI)
binding
Purpose
Binding site for repressor
Binding site for RNA polymerase
Gene encoding lac repressor protein.
Binds to DNA at operator and blocks
of RNA polymerase at promoter
Pi
CAP
Promoter for LacI
Binding site for cAMP/CAP complex
The LAC Operon: When lactose is present, it acts as an inducer of the
operon. It enters the cell and is converted to allolactose in a reaction
catalyzed by β-galactosidase. The allolactose form of lactose is the actual
inducer and binds to the Lac repressor, causing a conformational change
that allows the repressor to fall off the DNA. Now the RNA polymerase is free
to move along the DNA and RNA can be made from the three genes. Lactose
can now be metabolized.
When the inducer (lactose) is removed, the repressor returns to its original
conformation and binds to the DNA, so that RNA polymerase can no longer
get past the promoter. No RNA and no protein is made.
Note: RNA polymerase can still bind to the promoter although it is unable to
move past it because of the binding of LacI. That means that when the cell is
ready to use the operon, RNA polymerase is already there and waiting to
begin transcription; the promoter doesn't have to wait for the holoenzyme to
bind. We could say that the operon is primed for transcription upon the
addition of lactose. It is also important to remember that LacI does not bind to
DNA (Lac operator) with infinite affinity. Even in the absence of allolactose,
LacI will release the operator. Although another molecule of LacI will quickly
bound the operator site, a very brief period of transcription will result.
This allows the cell to continue to produce the small amount of βgalactosidase which is needed if the cell is to respond to lactose in the
environment (to make allolactose).
When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic
AMP is inhibited from forming. So when glucose levels drop, more cAMP forms.
cAMP binds to a protein called CAP (catabolite activator protein), which is then
activated to bind to the CAP binding site. This activates transcription, by increasing the
affinity of the site for RNA polymerase (makes the promoter stronger). This
phenomenon is called catabolite repression, a misnomer since it involves
activation, but understandable since it seemed that the presence of glucose
repressed all the other sugar metabolism operons.
Methods for Studying Regulation
Now think about what mutations in various elements of the Lac operon could
exist, and how we see these mutants as experimental scientists. First, here
are some notes about how we study the lac operon and some nomenclature
for how we describe the bacterial phenotypes that we see:
IPTG (isopropyl-beta-D-thiogalactoside) is a molecule that looks very
much like allolactose to the Lac repressor (LacI). Thus, this molecule can be
used as a gratuitous inducer, because it will induce the Lac operon by
altering the conformation of LacI so that it can no longer block the promoter,
but it is not a substrate for the lactose metabolism genes.
We can measure the amount of mRNA made on the lac operon (coding lacZ,
lacY, and lacA) by measuring the amount of β -galactosidase activity. This is
very easy, because β-galactosidase can be fooled into cleaving a colorless
substrate called ONPG into a yellow product called ONP. We can
quantitatively measure the amount of yellow in a spectrophotometer.
A constitutively expressed gene (denoted c) is never turned off. It is
making mRNA and protein all the time.
An uninducible gene is never turned on. An uninducible DNA binding site is
mutated so that it never binds its protein.
A super-repressor can be denoted s. This repressor always represses,
regardless of its regulation. For example, a LacI(s) mutant always represses
at the promoter regardless of whether or not allolactose is present.
Genotype
wild type
+
Phenotype
+IPTG
-IPTG
-
LacZ-
-
-
LacP-
-
-
LacO-
+
+
LacI-
+
+
LacI(s)
-
-
LacO-LacI(s)
+
+
Cis and Trans TEST
There are two different kinds of elements present in the lac operon's regulation. There
are DNA binding sites (LacP, LacO, Pi), and proteins (CAP, LacI, LacZ, LacY, and
LacA). When we study these systems, it is useful to be able to differentiate between
the two types of elements. For example, look above at the phenotypes of LacO and
LacI mutants. They are the same. How would we be able to distinguish a DNA-binding
site mutant from a mutant protein?
We can use techniques to see if a DNA sequence can act from afar on another DNA
sequence. If it can, then it is a diffusible protein. These sites are called trans-acting
sites, since they act from afar. If the site cannot act from afar, then it is a DNA binding
site that needs to be near other DNA sites (such as coding sequences) in order to
function. These sites are called cis-acting sites, since they need to be next to other
DNA to work.
In order to see if a DNA element is acting in cis or in trans to another DNA element,
we can do a test in which we insert a piece of DNA carrying element 1 into a cell that
already has a copy of mutated element 1 next to element 2 (we can then measure the
production of element 2 in our assays). If the inserted element can complement or
replace the function of the mutated element, it can be said to be acting in trans, since
it must diffuse off a plasmid or from another site in the DNA in order to be functional.
This, therefore, must be a diffusible protein. On the other hand, if the two functional
pieces of DNA must be adjacent to each other to be functional (acting in cis), then one
must be a DNA binding site affecting the other.
Therefore we can see that when we do the cis-trans test, any pair of DNA
elements that passes both the cis and trans test must be acting in trans (and
is therefore a coding region for a protein), and any pair that passes only the
cis test must be acting in cis (and is therefore a DNA binding site).
Strategies for Understanding Regulation
1. Find mutations that render the regulation uninducible or constitutive.
2. Decide by performing a complementation test if the mutants are
dominant or recessive.
3. If they are recessive, decide if the system is regulated by repression or
by activation. A recessive mutated activator has most likely lost function: the
system will become uninducible. A recessive mutated repressor has also lost
function, but now the system will have constitutive expression.
4. Decide if the elements of the system act in cis or in trans to each other:
are they diffusible proteins or DNA binding sites?
5.Construct a model.
There are a variety of additional mechanisms which prokaryotes have
developed to transcriptionally regulate gene expression. When we are
considering regulation of a system, we must always ask ourselves the same
question:
Why is the bacterium regulating this system?
When should these genes be turned on and when should they be turned off?
The Tryptophan Operon: A Repressor
When should the bacteria be transcribing genes for the synthesis of the amino acid
tryptophan? When levels of tryptophan in the cell are low, the bacteria must synthesize
the amino acid to allow protein translation to continue. However, if tryptophan is
abundant in the cell or can be scavenged from the environment, it is a waste of energy
for the bacteria to be synthesizing it.
The Trp repressor protein can bind to the operator of the Trp operon, which contains
the tryptophan biosynthetic genes. When tryptophan is in abundance, it binds to the
repressor and induces a change so that the repressor can bind to DNA. When
tryptophan levels are low, the tryptophan falls off the repressor, and the repressor
returns to its original conformation, losing its ability to bind to the DNA. The operator is
now free for RNA polymerase and transcription proceeds, making tryptophan
biosynthetic genes and replenishing the cell's supply of tryptophan.
This kind of feedback inhibition of transcription is very common.
The Histidine Operon: An Attenuator
The histidine operon functions in a slightly different way. At the beginning of
the operon there is a leader coding region with the following code and
corresponding amino acid sequence:
AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCUGAC
Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-Pro-AspWhen this sequence begins to undergo transcription, the mRNA leaves the
RNA polymerase complex and ribosomes bind onto it to start translation.
However, if there is little histidine in the cell, the ribosome stalls because
there are no aminoacyl tRNA's that are charged with histidine. This leaves a
long stretch of mRNA (for RNA polymerase is still transcribing) with no
ribosomes bound to it. The sequence of this RNA allows it to form a
terminator loop only when ribosomes are bound to it, at which point the RNA
is cleaved and the RNA polymerase stops transcribing the genes. Thus, the
terminator only functions when the ribosome is not stalled; that is, when there
is already plenty of histidine in the cell. The site at which the potential
terminator loop forms is called the attenuation site.
Note: Many amino acid biosynthetic operons are also controlled by some
form of attenuation. The tryptophan operon has attenuation control as well as
the repressor control described above.