Mod-01 Lec-01 Eukaryotic RNA polymerases and basal transcription factors

Friends, I am going to now start the first
lecture in this course, which is entitled eukaryotic gene expression, on basics and
benefits. Eukaryotic gene expression has become a very,
very important topic, and this century actually is going to witness a number of very important
key discoveries in this area. So, I have structured this course in such
a way that I am going to take you through a history of how many basic research has been
initiated in this area, maybe over the last 8 or 9 decades, and how things have progressed
and what stage we are; and during this entire course of research on basic aspects of gene
regulation and gene expression, people have also been in parallel being trying to understand
how we can translate some of these basic research findings for the benefit of mankind. So, the way I have structured this course
is to first tell you little bit about the basics of gene regulation; and once as we
start understanding some of the basic aspects of gene regulation, I am going to now mingle
this basic research into some of the important advances, or important key discoveries, that
has really led to some improvement of vaccine development, new therapeutics, new drugs,
new preventive tools, etcetera and so on so forth, so that you start appreciating why
it is important to understand gene expression, and how studying this gene expression is going
to help us to understand and find cures, or therapies, for many important diseases. So, the first lecture in this series, I am
going to give a very brief introduction about gene regulation in eukaryotes. We are basically going to talk about the workhorses
for gene expression, namely the RNA polymerases. These are the enzymes, which are actually
involved in synthesis of RNA from DNA. We are also going to understand how this RNA
polymerase is able to transcribe a gene, and how certain accessory proteins called basal
transcription factors actually help the RNA polymerase to transcribe a gene. This is what the crux of the today’s class. How RNA polymerase transcribe a gene, and
how certain protein factors called as basal transcription factors assist the RNA polymerase
to go and recognize what we call as a promoter elements and then regulate the expression
of various genes? Now, let us now try to understand that what
is the need for regulating gene expression. Now, all the cells in organism contain the
same set of genes. Now, whether you take a muscle cell or whether
you take a liver cell, whether you take a brain cell, the number of genes is more or
less the same in all the cases. In fact, this. For example, today, if I say the human genome
encodes about 30000 to 40000 genes, and every cell of our body has the same number of genes. But, what is very important, as far as the
gene regulation is concerned, not all the genes are actually transcribed in any individual
cell at any given time. The genes which are transcribed in a muscle
cell are different from the genes which are being transcribed in a liver cell or a brain
cell, and so on and so forth. So, the same complement of genes are not transcribed
in every cell of our body. So, this is what is called as a differential
regulation of gene expression. This is what is responsible for a liver to
look like a liver, or a muscle tissue to look like a muscle, or a brain tissue to look like
a brain, and so on and so forth. So, only fraction of the genes, or the total
complement of genes, are transcribed in an individual cell at any given time. So, it is this pattern of gene expression
that determines the structure, physiological function, and health of all cells and tissues
in the organisms. So, the differential gene expression is very
important and, although all the cells of our body has a same complement of genes, not all
genes are transcribed at same time. And only some genes are transcribed where
other remains silent; and this differential regulation is what is responsible for the
development and differentiation of an organism. Now, what is very important is that if this
gene expression does not take place in a normal manner, or aberrant gene expression can manifest
in the form a disease. A number of genetic disorders, including cancer,
can actually manifest if there are aberrations in this regulation of gene expression. So, what kind of genes have to be expressed
at a given time in a given cell is very important, and this is very finely regulated in our body. So, in fact, one of the biggest challenge
in the twenty first century is to understand how this differential gene expression is brought
about. Now, as far as eukaryotes is concerned, the
temporal and spatial expression of specific genes is vital to key life processes, such
as development, differentiation, and homeostasis. Especially, for those of you have studied
developmental biology, you will understand and appreciate, from the single cells I got
through a series of divisions, it develops into an adult organism; and each and every
step of this development and differentiation specific genes are expressed at specific time
points, and this spatial and temporal expression of these genes is actually responsible for
development of a fertilized egg into an adult organism. Even in the adult, once you become an adult,
specific gene expression in specific tissues and their proper regulation is responsible
for the correct functioning of the various tissues in our body. So, temporal and spatial expression of specific
genes is vital for all the cell life processes especially development, differentiation, and
the homeostasis. Now, gene expression is regulated primarily
at the level of transcription, and an understanding of the molecular basis for this regulation
continues to be a major challenge in twenty first century. So, what are going to now try to understand
is, what we know about gene regulation in the last few decades, and what are the challenge
that we are going to face in the twenty first century, and what are the key aspect that
we are going to understand in the next few decades. Now, let us start with some very basics. Now, the process of synthesis of RNA from
DNA is known as transcription, which I am sure all of you know; I do not have to elaborate
on this; and the key enzyme that is involved in the RNA synthesis is RNA polymerase. So, the enzyme which is responsible making
RNA from DNA is kind RNA polymerase. So, in today’s class, we are going to focus
entirely about this RNA polymerase and try to understand how this enzyme is going to
make RNA from DNA, and we have in the next 2 classes we are going elaborate little bit
more about what kind of transcription factors are actually helping the RNA polymerase to
transcribe by a gene. Now, in eukaryotes, there are 3 distinct types
of RNA polymerases, of which the RNA polymerase, which transcribes protein coding genes or
which makes messenger RNA, is called RNA polymerase 2. So, the transcription of genes by RNA polymerase
2 results in the synthesis of messenger RNAs in the nucleus. Translation of these mRNAs in the protein
by the protein synthesizing machinery in the cytoplasm results in the synthesis of proteins. So RNA polymerase 2 in eukaryotes is primarily
responsible for making proteins. So, the polymerase 2 goes and binds the promoters
of protein coding genes. It makes messenger RNA, and this messenger
RNA comes into cytoplasm gets translated into proteins. What there are 2 other RNA polymerases. We will talk about it little bit later. Now, coming back to the other point, the synthesis
of this protein in cells is a highly regulated process. I told you, in a sense, not all the proteins
are made in the all the cells of our body. Specific proteins are made in specific cell
types, and we need to understand how this regulation is brought about. Although RNA polymerase is there in all the
cells, and all those genes are there in the cells, the RNA polymerase does not transcribe
all the genes, all the time, and all the tissues. It is very selective. Now, when a particular protein is to be synthesized,
the specific gene coding for the cognate mRNA has to be transcribed. This is where the regulation is comes into
the picture. Only those proteins needs to be synthesized,
and only those genes need to be transcribed the RNA polymerase, whereas other genes whose
proteins are not required, those genes should not be transcribed by RNA polymerase. This is the molecular basis for differential
gene regulation. So, as we have for a very basic definition
of what we are going to study today, the mechanism by which transcription of genes is activated
or inhibited is referred to as gene regulation. This is the primary objective of this entire
course; trying to understand how this gene regulation is brought about, how this RNA
polymerase is selectively able to activate the expression of certain genes and inhibit
the expression of other genes, is what is going to be the crux of this particular course. Now, before we go to the eukaryotes, let us
understand, gene… the gene regulation has been extensively studied in prokaryotes because
prokaryotes are very simple organisms. They are unicellular organisms and they have
much less complicated, much less complex than eukaryotes. So, in the last century, early part of the
last century, a lot of effort has gone in to understand how gene regulation is takes
place in prokaryotes. So, let us take bacteria, which are simplest
form of prokaryotes. Bacteria respond to external condition by
regulating levels or the activity of certain key enzymes. For example, if a bacterium is grown in a
medium containing lactose, normally glucose is the most preferred carbon source because
the simplest form of sugar and it can be readily metabolized, and very easily energy can be
generated without expending energy. But, suppose there is no glucose in the medium
and there is only lactose, and lactose as you know, is a disaccharide comprising of
glucose and galactose, and therefore, if the organism has to metabolize lactose, how does
it react? Now, it is what does the cell has to do, if
it has to know metabolize lactose instead of glucose. First, it has to import the lactose from the
medium, and then it has to cleave the lactose to glucose and galactose, because as I said,
galactose is disaccharide; it has to be first broken down into glucose and galactose, and
then the galactose has to be converted into glucose, and then glucose then enter by…
enters the metabolism like glycolysis, Kreb cycle, and so on so forth, and energy is generated. Now, bacteria, therefore, have to produce
the enzymes required for lactose metabolism only when exposed to lactose; it makes common
sense. When there is no lactose in the medium, why
should the organism make enzymes that is required for import of lactose, or to cleave lactose
to glucose or galactose, or convert galactose to glucose? It is totally a waste of energy. Therefore, bacteria need to express the genes
that code for the enzymes involved in these processes only when lactose is present in
the medium. Now, in the early part of the last century,
the twentieth century, allosteric rare variation, allosteric regulation of enzymes by smaller
molecules was very well studied, and enzymology always had an upper hand compared to molecular
biology and understanding gene regulation. Therefore, researchers were studying, actually,
how enzymes function and how small molecules are metabolized, and so on and so forth. But, however, how actually enzyme production
is actually regulated by small molecules were not very well understood. This is, I am talking sometimes early 1950s
and 1960s. So, while you very well know how small molecules
are actually catalytically activated by enzymes, and how enzymes metabolize many of the small
molecules, is rather well understood. How exactly the small molecules activate expression
of genes leading to synthesis of enzymes is not very well understood in the early part
of the last century. A series of investigations, especially those
by the Jacob and Monod, who actually won the Nobel prize at, later revealed that in bacteria,
genes involved in specific metabolic of biosynthetic pathways are grouped together as operons and
their expression is coordinately regulated by small molecules. Now, I am not going spend too much time trying
to explain to you prokaryotic gene regulation. In fact, I have organized this course with
the basic understanding that you people have already studied how gene regulation takes
place in, in prokaryotes. The concept of operons, and how e coli RNA
polymerase goes and binds to promoter sequence and activate transcription. These, I am sure, you must be having some
basic knowledge. So, I am not going to tell too much about
the operons. Just supposed to know that, in the case of
bacteria, genes involved in specific metabolic pathways or biosynthetic pathways are organized
in the form of operons, and therefore, these genes are coordinately regulated. This is what we need to know. Now, in a bacterial operon, the binding of
RNA polymerase to its cognate promoter is regulated by an operator repressor mechanism. I am sure this also you have very well understood,
and you have studied some of the basic textbooks on how actually a something like a lac operon
or trp operon is regulated. I am going to give a very brief introduction
about it, just to recapitulate what already about bacterial gene regulation. I am sure you are also aware there are actually
2 types of operons in bacteria. One is inducible operon, another is repressible
operon. Now, the best example, I am sure most of you
have studied it for inducible operons, is— In a inducible operon, the ability of an RNA
polymerase to bind to the promoter and transcribe the structural genes located downstream of
the promoter is controlled by an operator sequence. So, you have an operator sequence linked to
a downstream promoter sequence, and usually a repressor molecule goes on binds in the
operator and prevents the RNA polymerase from binding, and that is what I have written here. The function of an operator is controlled
by a repressor protein, which binds to the operator and prevents RNA polymerase from
binding the promoter. So, this is how the operon is not transcribed
or it is kept in a silent mode. So, as long as the repressor binds the operator
sequence and prevents the RNA polymerase from binding the promoter, the operon is not functional,
and therefore, these genes are not transcribed. And therefore, enzymes of that particular
pathway is not synthesized. Now, the repressor protein is kept in an active
state by an inducer molecule, which usually is the substrate for the enzymes encoded by
the structural genes of the operon. For the best example is, for example, lactose. I am sure all of you have studied. When there is a lactose in the medium, the
lactose goes and binds the lac repressor and prevents the lac repressor from binding to
the operator, and therefore, RNA polymerases can now go and bind to the promoter sequences
and transcribe the structural genes. Now, all these genes now result in the synthesis
of enzymes, which will then import lactose in large amounts, cleave the lactose into
galactose and glucose, and then the galactose is further epimerized into glucose, and then
glucose is further metabolized and derive energy. You also have examples of a repressible operon. For example, the tryptophan operon wherein,
it is also similar to inducible operon, except that the repressor, in this case, is activated
by a chemical substance called co-repressor, which is usually the end product. Now remember, inducible operons usually are
involved in metabolic pathways, in catabolic pathways like lactose degradation and so on
and so forth, whereas repressible operons are usually involved in biosynthetic pathways
like, for example, tryptophan biosynthesis. It is actually the trp repressor, which is
the end product of the tryptophan biosynthesis that acts as a inhibitor of trp operon. Now, I am not going to talk a little bit more
about the operons, because I am sure all of you aware of it. But, one point I want to make, before I switch
to eukaryotic gene regulation, is that not all the genes in in prokaryotes are always
in the form of operons. There are many genes which are have their
own individual promoters, and then you also question— do all these genes which have
their own individual promoters, are they all regulated in the same manner? They are all expressed at the… Do they make same levels of RNAs? The answer is no. So, there is differential gene regulation
of these individual genes. That means, there has to be regulatory mechanisms
of these genes, which are not organized into the operon. So, what I want to now stress, may be couple
of minutes, is, how about the regulation of e coli genes and promoters, which are not
organized into operons? So, there are a number of genes which are
not organized into operons and such genes also contain their own promoters. And since all the such genes are not expressed
the same level, we need to understand how differential regulation is brought in this
case. Now, this is primarily achieved by the variation
of the smallest subunit of bacterial RNA polymerase, known as the sigma factor. I am sure all of you are aware that the e
coli RNA polymerase or the bacterial RNA polymerase is a tetramer, which consists of 2 alpha subunits
and 2 beta subunits, and it also has a very small subunit called sigma subunit. Now, the sigma subunit is actually responsible
for gene regulation in the case of eukaryotes, and these genes which have their own individual
promoters, these genes are differentially expressed by, actually, by synthesizing different
sigma factors. Now, it turns out, if you look at the e coli
promoters, the among the in the RNA polymerase holoenzyme when I say RNA polymerase holoenzyme,
which means, the RNA polymerase core enzyme, which consists of the 2 alpha and the 2 beta
subunits, and the sigma subunit. It is the sigma subunit which actually recognizes
specific promoter sequences in the upstream region of the genes, and these are all actually
called as the minus 35 sequence and the minus 10 sequence of the Pribnow box. Now, the consensus sequence for the sigma
subunit is the Pribnow box usually contains TATAAT sequence, and a minus 35 box usually
contains TTGACA sequence, and these are the 2 sequences which are actually recognized
by sigma subunit of RNA, e coli RNA polymerase. But, it turns out, not all the e coli genes
contain exactly the same sequence. There are minor variations within the sequence,
and I just gave here four examples. There are four different genes where you can
see, there are minor variations in this consensus sequence. It turns out, these minor variations in this
minus 35 and minus 10 sequence is actually responsible for differential regulation of
gene expression in bacteria. Now, there are certain sigma factors which
will only recognize this minus 35 and this minus 10, but not this minus 35 and minus
10. So, only those genes, which contain this particular
type of sequence, will be activated by that particular sigma factor. So, by minor variations in minus 35 and minus
10 sequence, different sigma factors can bind different promoter sequences and activate
or repress different genes. I just give an example here. For example, if for example, let us say the
e coli has to activate, has to now grow, and there are about 1000 genes which now have
been activated for the e coli cells to grow. And what does it do? The e coli now makes a specific sigma factor
called RpoD, and this RpoD goes and binds to specific minus 35 and minus 10 sequences
of these 1000 growth related genes, and activates all these genes. Whereas, if suppose, there is a starvation,
there is a nitrogen regulation, there is a stress response, there is a nitrogen starvation,
or there is a stress response; and let us say, there is above fifteen genes which now
need to activated, a specific sigma factor called RpoN now goes and binds to these minus
35 and 10 sequences of these genes and activates the transcription of these genes. So, by synthesizing different sigma subunits,
which have the ability to recognize different minus 35 and minus 10 sequences, e coli can
bring about differential regulation of gene expression. Now, so, what I told so far is that in prokaryotes,
gene regulation allowed them to respond to their environment efficiently and economically. Either the genes can be grouped in the form
of operons as I told in the beginning, or there are variations in the minus 35 and minus
10 sequences recognized by the sigma factor, and using these variations, different genes
can be transcribed by using different sigma factors. This is how differential gene regulation is
brought about in bacteria. There are other variations, which we will
not dwell about at this time. So, in the e coli, a single RNA polymerase
with the help of different sigma subunits can bring about differential gene regulation,
together with specific the activators or repressors, in the case of operons. Now, now let us come to eukaryotes. Now, in the case of eukaryotes, in addition
to these environmental responses or environmental stress signals, gene regulation became essential
for the control of a number of cellular processes, such as cellular differentiation during development,
immune responses, tissue-specific functions, and so many other processes. Because the eukaryotes become more and more
complicated, you have various tissues and you have developmental band, which is very
different. There are many proteins have to be made, and
they have to be regulated properly, and the nervous system is much more complex. So, there are all kinds of complications. So, to take care of all these things, the
gene expression has to be regulated in much more complicated manner. Then what does happen in the case of e coli? So, e coli is able to rule just 1 e coli RNA
polymerase and a bunch of sigma factors, but this alone is not sufficient. When it comes to eukaryotes, you require lot
more complex regulatory machines. So, to meet this complexity of this eukaryote,
as a first step, the number of RNA polymerases were first increased from 1 to 3 in eukaryotes. So, while in the case of e coli, just 1 RNA
polymerase was able to do everything, in the case of eukaryotes, says it, the 1 RNA polymerase
became 3 to take care of this complexity. So, let us now spend some time to understand
what are these eukaryotic RNA polymerases, and how they have evolved. So, as I told you, in the case of bacteria,
the core RNA polymerase consists of 2 alpha subunits and 2 beta subunits and with the
help of different sigma factors, it can bring about a whole bunch of gene regulation. Now, when it comes to eukaryotes, instead
of one RNA polymerase, now you have got 3 different RNA polymerases, which are usually
designated as polymerase 1, polymerase 2, and polymerase 3. Now, I am going now spend some time on the
history. Now, it is always nice to remember some historical
aspects and let us see what kind of effort actually went to understand the various eukaryotic
RNA polymerases, and biochemistry plays a, played a very important role in a understanding
the function of various eukaryotic RNA polymerases. So, the first step, you want to understand
the function of an enzyme you have to now purify. You have to, because when this when a mixture
of protein, you cannot really study and then understand the function of a particular enzyme. RNA polymerases are nothing but enzymes. These are enzymes which make RNA. So, as is true for any enzymes, if you want
to know, study, and understand how these RNA polymerase function, the first thing you have
to do is to purify the eukaryotic RNA polymerases. But, there was a problem. Well, in the case of mammalian cells or in
eukaryotes, the RNA polymerase was tightly bound to chromatin; know in the case of e
coli, you do not have a very well-organized chromatin structure. But, in the case of eukaryotes, you have a
very compact chromatin structure, which is organize in the form of a… I mean, inside the nucleus, and this RNA polymerase
is very tightly bound to chromatin as a result of its engagement to active transcription;
it first became essential to develop new methods to solubilize RNA polymerase and remove the
interfering DNA and histones. Because DNA is tightly bound with histones
and is organized in the form of chromatin, and if you want to study, understand, how
RNA polymerase regulate, and you want to purify RNA polymerase from this, first you have to
dissociate histone and DNA and then remove the RNA polymerase; then only you can study
function. So, a lot of effort went into under to purify
this RNA polymerases, or isolate this RNA polymerase from this DNA and histones. So, the key, or the 3 key steps which were
actually discovered to find the purification of RNA polymerases— first, you have to disrupt
the nuclei, and then dissociate the histones from DNA using very high salt. Now, histones are positively charged and DNA
is negatively charged. Therefore, their association is very tight
and therefore, you have to first dissociate the histones and DNA using very high salt
concentration; something like 2 molar NaCl, and once the histone dissociates from the
DNA, then you break the DNA and dissociate RNA polymerase by sonication, and then you
selectively precipitate the DNA protein complex. And now, you have the RNA polymerase with
other proteins in the solution, and that is what your starting material for purifying
the RNA polymerase. So, this soluble enzyme preparation, which
is devoid of DNA and histones, was then used to purify RNA polymerase. Now, I want to spend some time on a very important,
very key experiment, which was actually described in nature by Robert Roeder’s group in 1969,
which is how exactly the 3 RNA polymerase were discovered in eukaryotes. Now, what basically it is, once you have a
soluble enzyme fraction which contained RNA polymerase and many other soluble proteins,
the next step is to you have to purify. Now, protein purification as you know, is
done conventionally by protein chromatography techniques. You are aware that proteins can be separated
based on their charge, or proteins can be separated based on their mass or molecular
weight. Now, based on charge it is called ion exchange
chromatography, and if you want to separate based on their mass or molecular weight, you
can use what it is called as a gel filtration chromatography. So, what these people did in the late 1960s? They took the soluble enzyme preparation which
is devoid of DNA and histones, which are either prepared from sea urchin embryos, or yeast
cells, or drosophila embryos, or hela cells, and so on and so forth; and then put it on
a ion exchange column. For example, in this case, DEAE Sephadex column. Now, what happens when you put this mixture
of proteins on a DEAE column? Proteins bind to this column depending upon
their charge, and they can be… proteins may be separated based on their charge, and
proteins which do not bind this column, they first elute in the void volume, and then you
get different, various other proteins. And once you are sure the proteins can be… Once the proteins are bound to this ion exchange
column, they can now be eluted by increasing the salt concentration. So, depending upon the affinity of the proteins,
that is, proteins which have, for example, very low affinity to the ion exchange resin
will elute first with very low salt concentration, whereas proteins which have very high affinity
for the ion exchange resin will get eluted later. So, we will get… so if you now start collecting
fractions with increasing salt concentration, you can collect various proteins in different
fractions, and you can see, accordingly, you will get what is called as a elution profile
of the protein. Now, you take these different fractions of
these various proteins, which are now separated, based on their charge, and then ask the question—
which of these fractions actually contains RNA polymerase activity? When they did this experiment, they found
there was no RNA polymerase activity in this total, in this major protein peak, but the
RNA polymerase activity was actually present in three distinct peaks, which I have shown
here, in the red. So, the total protein peak is different, and
the RNA polymerase activity peak is different, and surprisingly, they got 3 distinct peaks
of RNA polymerase activity, which they designated as polymerase 1, polymerase 2, and polymerase
3. Now, this they have got in a very reproducible
fashion. Every time they took this soluble enzyme extract
and put it on a ion exchange column, they always got 3 peaks of RNA polymerase activity,
clearly saying that this is not an artifact. There are, in fact, some 3 different kinds
of RNA polymerase activities in these cells, and they probably have some very important
key differences. Now, one important difference that I actually
found out, is that there is a fungal toxin called alpha amanitin, which was isolated
from a fungus called Amanita phalloides, and this alpha amanitin binds very tightly to
RNA polymerase 2 and blocks transcription elongation. So, if you add this alpha amanitin to cells,
it will go and bind to RNA polymerase 2 and prevent transcription by RNA polymerase 2. Very interestingly, this alpha amanitin does
not bind to RNA polymerase 1, and it requires very high concentration to bind to RNA polymerase
3. So, of the 3 RNA polymerase that they identified,
the RNA polymerase 1 is insensitive to alpha amanitin, whereas RNA polymerase 2 is highly
sensitive, whereas RNA polymerase 3 requires slightly, for example, ten microgram per ml
of alpha amanitin is required to inhibit the activity of RNA polymerase 2. So, by just, say, simply look at this sensitive
of this RNA polymerase to the alpha amanitin, you demonstrate there were actually three
distinct types of RNA polymerase— alpha amanitin insensitive RNA polymerase 1, highly
sensitive RNA polymerase 2, and an intermediately sensitive RNA polymerase 3, which was highly
present in the eukaryotic cells. By using this particular mechanism, or using
this particular observation, what they by simply monitoring the alpha amanitin sensitivities
of specific transcription events by endogenous RNA polymerase in the isolated nuclei, it
was actually demonstrated that rRNA is synthesized by RNA polymerase 1, whereas messenger RNA
synthesized by polymerase 2, and the 5S and tRNA is actually synthesized by polymerase
3. That means, what you do, you take the nuclei,
and now if you add alpha amanitin, and you find that ribosomal RNA is still being synthesized,
which means, that since we already know polymerase 1 is actually responsible for making ribosomal
RNA and polymerase 1 is not to alpha amanitin, it meant that ribosomal RNA is actually being
made by the alpha amanitin insensitive RNA polymerase 1. Whereas in the same nuclei, if you now add
a very small of alpha amanitin, no mRNA could be synthesized, and since in the… from in
vitro experiments we already know the RNA polymerase 2 peak is highly sensitive to alpha
amanitin, it concluded that, for synthesis of messenger RNA by this RNA polymerase 2
which is highly sensitive to alpha amanitin, RNA polymerase 2 is responsible for mRNA synthesis,
and so on and so forth. Now, today we actually know that, not only
there are these distinct RNA polymerases, they actually have very very specific functions,
and we know, that the RNA polymerase 1, which actually makes the ribosomal RNA, is actually
present in nucleolus, whereas the other 2 RNA polymerase, both 2 and 3, are present
a nucleoplasm; one of them actually responds for making messenger RNA and snRNAs, and the
RNA polymerase 3 is actually responsible for synthesizing transfer RNA, 5S RNA, and several
other small nuclear RNAs. So, this is how the 3 RNA polymerase were
actually discovered, now. So, once you started purifying the identify
the RNA polymerase activity by using series of chromatographic steps, the RNA polymerases
were kept on… were being purified, and once they have got a pure RNA polymerase 2 activity,
and when they know, subject to what is called as SGS polyatomic gel electrophoresis, they
found this pure RNA polymerase actually has a number of subunits. So, you can see the equal RNA polymerase core
enzyme had only 2 alpha and 2 beta subunits, whereas if you now take the purified RNA polymerase
2, it had at least 9 different subunits or 12 different subunits, as I showed here, and
each of them has a different molecular weight. So, this clearly told that the eukaryotic
RNA polymerase is much more complex than the prokaryotic column RNA polymerase and it has
many more subunits than the… its prokaryotic counterpart. Now, let us now trying to see what are the
commonalities and what are the differences in the RNA polymerases between the various
RNA polymerases in eukaryotes. Now, in the case of yeast cells, which is
also an eukaryote, all the 3 RNA polymerases have 5 core subunits, which have homology
with the some of the subunits of e coli RNA polymerases; clearly telling that they are
all originated from the e coli. E coli RNA polymerase is the ancestor for
evolution of this eukaryotic RNA polymerase. Now, the RNA polymerase 1 and 2 contain the
same two non-identical alpha-like subunits. Polymerase 2 has two copies of a different
alpha-like subunits. The four subunits which are present are common
for all the three RNA polymerases, that is, of the various subunits of each of these RNA
polymerases. Four are common for all the three RNA polymerases,
and each of these RNA polymerase have at least 3 to 7 unique smaller subunits. Now, this is very important, what I am telling
you. Now, the largest subunits of RNA polymerase
2, the largest subunit of RNA polymerase 2 means, that is, the 100 and 90 kilo dalton
Rpb1. This largest subunit of RNA polymerase 2 has,
what is called as a C-terminal domain, where in the next couple of classes this is going
to be very important, and I am going to tell you how important is the C-terminal domain
of this large subunit of RNA polymerase 2. Remember, the largest subunits of RNA polymerase
2 contain a very important part called as C-terminal domain or called as a CTD. Now, what is so unique about the CTD, both
in yeast as well as humans, and all other mammalian cells, this CTD contains what is
called as a YSPTSPS repeat motif. As you know, Y is a tyrosine; S is serine;
proline, threonine. So, tyrosine, serine, proline, threonine,
serine, proline, serine; and as you know, serine and threonine are the residues which
can be phosphorylated. So, the C-terminal domain has a highly serine
and threonine rich motif, and in the subsequent process I will tell you that phosphorylation
of the serine and threonine residues in the CTD plays a very important role in regulating
the activity of RNA polymerase 2. So, just remember; the largest subunit of
RNA polymerase 2 contains a C-terminal domain, which is has a number of repeats containing
serine and threonine residues, and at a later stage I will show you that phosphorylation
of this and this plays a very important role in regulating the activity of RNA polymerase. That is what I have written here. Now, so what I told you so far, is, early
studies of the mammalian RNA polymerases provided the first indication that eukaryotic transcription
machinery is far more complex than that existing in prokaryotes. I think, you will now agree, because of e
coli you just had 4 subunits of the core enzyme and you had may be a, a dozen or so of dozen
or 2 dozens of sigma subunits, and with that, e coli was able to manage differential gene
regulation. But, when we came to eukaryotes, the number
of RNA polymerase itself was increased to 3, and as we proceed further, I will now tell
you it is not just the increasing number of RNA polymerase 2 subunits, RNA polymerase
subunits, but even a number of other accessory proteins are also essential in order to accurately
initiate transcription by RNA polymerases in the case of eukaryotes. Now, so what I told you is that, historically,
around the late 1960s, people began to purify RNA polymerases from various eukaryotic cells,
and they still did not understanding that these RNA polymerases are much more complex
and they contain multi-subunit processes. And as one group started purifying this RNA
polymerase and try to understand how these RNA polymerases are regulating, and isolate
and characterize what are the subunits, and so on and so forth, there was also another
group which started looking at what are the sequences to which these RNA polymerases go
and bind and activate gene regulation. So, 2 distinct approaches were being simultaneously
being followed to understand gene regulation in eukaryotes. In one case, determination of DNA sequence
requirements for initiation using cell-free transcription and transfection assays. I would elaborate little bit on this later. That means, you need to identify how it is
exactly that even the eukaryotic RNA polymerase goes and binds the promoter sequence. Like in the case of e coli, I told you, the
sigma factor binds to this minus 35 and minus 10 sequence, and that is what is responsible
for differential gene regulation. So, are there similar elements in the case
of eukaryotic promoters, and what are these promoter sequences, and how these promoter
sequences function in vivo by using what are called transfection assays, or what are called
as a cell-free transcription assays, where you actually make cell-free extracts from
various cells, and in a test tube, you now add DNA templates, you add various ribonucleotides,
and you add ATP and ask the question, whether now the purified RNA polymerase can initiate
transcription accurately in a test tube; that this is what is the in vitro or cell-free
transcription system. So, 2 approaches were being followed. On one hand, people were trying to purify
RNA polymerases from various cell extracts and what kind of subunit structure, and so
on and so forth; on the other hand, people were asking the question, if we now take this
purified RNA polymerases and add to a test tube, which contains the promoter region of
a gene, and now if you add all the factors which are required for it, such as the template
DNA containing promoter sequence, if we now include all the energy, and if you require
all the NTPs, will this RNA polymerase be able to initiate transcription? That is, the unless RNA polymerase goes and
binds to specific sequences in the promoter, it cannot initiate transcription, and what
are these sequences? Are the sequence similar to what is there
in the prokaryotic promoters in the eukaryotes? So, isolation and characterization of unknown
protein factors that are involved in the accurate initiation of transcription by RNA polymerases
has been carried out on one hand, and when they add all these purified RNA polymerases,
they are putting these RNA polymerases in a cell-free system on a transfection assay
and ask the question— how these RNA polymerases are able to initiate transcription? Now, such studies came into, gave a very important
and interesting observation. It turns out, when people started looking
at the sequence of this promoter region, especially, the region is about, within about 50 bases
from the transcription start site of eukaryotic promoters, they came up with a very interesting
observation. If you now look at some this sequence, you
can see, if you take, for example, the numbers I have given is for about is a percentage. If you take, for example, 100 genes and look
at the promoter regions of this 100 genes, for example, in this particular position,
in 97 out of 100 genes, always it was an A. Whereas in this position, for example. Similarly, in this particular position, in
85 out of 100 genes, there was again a A, whereas in this particular position, 81 percent
of the genes had a T. Here, 97 percent of the genes had a A, 91 percent here had a T
residue, 85 percent had a A residue, 63 percent had A or T, 88 percent of genes had a A residue. So, which clearly tell that there is some…,
this TATATA motif seems to be highly conserved in the promoter region of a majority of protein
coding genes in eukaryotic genes, eukaryotic promoters. So, this is now being now recognized as what
is called, say, TATA box. As we go along, you realize that this TATA
box is the place where actually transcription RNA polymerase and other factors actually
go and bind, and this assembly of RNA polymerase in and around TATA is actually responsible
for accurate initiation of transcription in the RNA eukaryotic promoters. So, the first major finding about the organization
of eukaryotic promoters is the presence of a highly conserved sequence called TATATA
motif. This is known as the TATA box, and this TATA
box seems to be conserved in a number of eukaryotic promoters. So, this is the first important finding from
the analysis of various genes transcribed by RNA polymerase 2. Now, so in the late seventies, people actually
started doing experiments, not only in the cell-free systems containing DNA templates,
but they also started using chromatin templates. And such studies, for example, by Carl Parker’s
group actually showed that in the case of RNA polymerase 3, it actually can transcribe
the 5S RNA genes in purified chromatin from immature oocytes, but not in total cellular
or cloned 5S DNA templates. That means, the RNA polymerase 2 actually
can transcribe the 5S or polymerase 3 can transcribe 5S RNA only in a chromatin-based
template, but not in DNA template devoid of chromatin, just plain, naked, DNA template. Which actually told that, for RNA polymerase
to transcribe accurately genes, some factors in the chromatin are also required. Just naked DNA alone is not sufficient. Some chromatin templates containing other
accessory proteins are actually extremely required. So, the… So, this expert actually suggested that there
are chromatin bond factors which are actually essential for RNA polymerase 2 actually transcribe
some of these genes. There are many other experiments which are
carried out. I will give a key references at the end of
the presentation. You can go through some of these experiments
and read a little bit more on how people actually started fractionating extraction, start putting
this extract in the cell-free system, and started identifying what kind of factors are
actually required for RNA polymerase to accurate initiate transcription. I am just giving one example here. For example, fractionation of nuclear extracts
and identification of factors essential for accurate initiation of transcription. So, as I said, they took the soluble nuclear
extract first, put it on a, for example, a phosphocellulose column, and eluted the protein
with varying salt concentration; for example, here, the point 35 molar NaCl, point 6 molar
NaCl, 1 molar KCl. Now, you now take these proteins which got
eluted from the point 6 molar KCl fraction, and again put it on a DEAE cellulose column,
and then proteins bound to the DEAE cellulose column, again you start eluting at different
salt concentration. And again, take this fraction, put on another
DNA cellulose column, start eluting at different salt concentration, and you now start looking
at each one of these fractions, and see which one of this fractions contain protein factors
which are essential for RNA polymerase to accurately initiate transcription. Now, I want to describe here a very important
assay, which is acts known as the runoff transcription assay. What actually you do in this assay is that
you take a DNA template which contains the promoter sequence, and then ask the question—
how RNA polymerase will go and bind to this promoter sequence? And it binds, you have what is called as a
linear template. Let us say, for example, a 400 base pairs
downstream of a promoter. So, when RNA polymerase binds and then starts
transcribing the gene it will fall off at the end of this 500 base pairs, and you will
get a 500 base pair RNA. So is called a runoff transcript. So, this what I have shown here. If you now take some of this chromatographic
fractions, and then add it to RNA polymerase, some of these chromatographic fraction, for
example, a, c, and d. That is, a, c, and d was able to support accurate
transcription by RNA polymerase 2, but for example, if you now add a, add only a and
e, it does not support, give any runoff transcript. So, these… By using these kind of runoff transcription
assays, people started identifying which chromatographic fraction actually contains those protein components,
which are actually essential for RNA polymerase to accurately initiate transcription. At the same time, another important study;
as people started identifying some of these protein factors which are actually helping
the RNA polymerase to bind and accurately initiate transcription, people also start
using what is called as the super shift assays. Now, here, what you do, once you purify each
one of these transcription factors, which are actually as protein factors which are
helping the RNA polymerase to accurately initiate transcription, you would now start asking
the question— are these protein factors interacting with each other or not? So, what you do is a very important. We have a shift, what is called as a gel electrophoresis
mobility shift assay, where you take the promoter DNA fragment, like the containing the TATA
sequence, radio label it, and now you add, for example, a protein factor which is, which
you have purified in the, as I have showed in the last slide. When this protein factor binds this DNA sequence,
it causes a mobility shift, and you can see the probe will get shifted here. Now, you add another protein component, for
example, when this is just the naked radio labeled promoter DNA alone, which moves here. Now, when this DNA binds to protein, it causes
the mobility shift and you get a complex here. Now, if one more protein is binding to this,
now you will see a further shift in the mobility and this indicates that, what it tells you
is that, by one by one, number of protein factors can actually assemble over the promoter
sequence. By doing this, generally, people have actually
demonstrated there is a very sequential assembly of protein factors on the promoter DNA template. So, this is a key factor which actually demonstrated
that certain fine intermediate complex in the transcription initiation were actually
identified by RNA polymerase 2. Based on this thing, they proposed a model
by which sequential assembly of transcription factors on the promoter sequence is actually
responsible for accurate initiation of transcription by RNA polymerase 2. So, between 1980 and 1990s, studies from various
laboratories have actually established that accurate transcription initiation by RNA polymerase
2 requires polymerase 3, requires 2 transcription factors called as TF3C and TF3B, which contain
at least 9 distinct polypeptides. So, using runoff transcription assays and
also using these kinds super shift experiments, people have identified, at least, there are
2 protein factors which are actually required for RNA polymerase 3 to transcribe tRNA and
5S rRNA genes. Similarly, in the case of RNA polymerase 2,
you require a number of protein factors which are named as TF2D, TF2A, B, E, F, and H, and
which contains about, in all put together, they are about 32 different polypeptides,
which actually are essential for RNA polymerase to accurately initiate transcription in the
case of protein coding genes. In the same way, several polymerase 1 factors
are also identified, and all these factors were found to be structurally and functionally
very distinct. So, and they also found out, whether you purify
these transcription factors from the yeast, or drosophila, or mammalian cells, or sea
urchin cells, they all seem to be highly conserved. So, which clearly told you that these protein
factors which help the RNA polymerase to accurately initiate transcription, they are highly conserved
from yeast to man. That was the outcome of the various research
that took place between 1980s and 1990s. So, I have just given you the summary, here,
of the various research effort that went into to identification of all these key accessory
proteins that help the RNA polymerase from for transcribing RNA protein coding genes. For example, you have what is so called as
a TF2D, which means transcription factor 2D. One of the important components of transcription
of 2D, what is called as a TATA binding protein. This is the one that actually recognizes the
TATA box and brings responsible for specific assembly of a RNA polymerase similar in the
TATA box. Each contains about, the TBP contains about
a 38 kilo dalton protein. Its main function is to recognize the core
promoter sequence, that is the TATA box, and also now, then that would be next transcription
factor called TF2B. There is another; the TF2D, also, in addition
to TBP, contains what are called as TBP associated factors. There are about 12 of them with a molecular
weight ranging from 15 to 200 and 50 kilo daltons and their job is to actually help
the RNA polymerase in transcription activation, and also in promoter reorganization. Similarly, you have protein factors called
TF2A, TF2B, TF2F, and TF2E, and each of them has a very specific function in the initiation
of transcription, especially, the TF2H. For example, TF2E is actually involved in
the recruitment of what is called as TF2H, and the TF2H actually has what is called as
a helicase activity, and this helicase activity is actually responsible for modulating the
activity for RNA polymerase 2. And I told you in the in the previous 2 slides
that RNA polymerase 2 actually contains what is called as a helicase domain, sorry, carboxy
terminal domain, which is rich in serine and threonine routines, and it is the TF2H which
actually phosphorylates it is carboxy terminal domain of the RNA polymerase, and it actually
responsible for converting what is called as initiation the complex to a elongating
complex. So, it is almost like a train that, if the
train is there in the station, and the train wants to move, the guard has to wave his green
flag, or a green signal has to come and say. Only then, the train starts moving. The same way, if the RNA polymerase has to
leave the promoter region and start transcribe the gene, it has to receive some specific
signals. And when that when does, when does that happen,
the transcription initiation can be take place when the cell has to that all the components
require for transcription initiation is actual present in the cell. For example, it has all the required ribonucleotides,
all the factors required for transcription is actually assembled, and the entire RNA
polymerase holoenzyme has actually assembled in the promoter region, and then, when we
have all the ribonucleotides, the TF2H now comes and phosphorylates this carboxy terminal
domain of the RNA polymerase 2, and that is the signal for RNA polymerase to now leave
the promoter, or leave the station and stop moving. So, remember, the initiation RNA polymerase
2 in the initiation complex is non-phosphorylated, whereas in the elongating RNA polymerase 2
the carboxyl terminal domain is highly phosphorylated, and this phosphorylation is actually done
by one of the accessory proteins required for transcription initiation, namely, the
TF2H. It also has other helicase and kinase activities,
and so on so forth. So, what I told you so, far, is that there
is a very ordered assembly, or what is called as a pre-initiation complex formation in an
eukaryotic protein coding gene promoter. You have, for example, the RNA polymerase
2, which itself contains up to 12 subunits in the case of yeast, and this polymerase
2 first has to associate with the protein factor called TF2F, and this polymerase 2
TF2F is then goes and binds to the promoter region. And in this promoter region, first the TF2D,
which contains the TATA binding protein, comes and binds. Then, it recruits TF2B and TF2A, and this
is actually called as a formation of a DAB complex. So, once the DAB complex is formed, that is
the signal for the pol 2 and F and come and join the promoter region, and once the DAB
F and pol 2 complex is assembled, now, the TF2H comes and binds here, and it makes sure
that everything is ready. Then, it phosphorylates the carboxy terminal
domain of RNA polymerase 2, and then the polymerase 2 starts moving and starts the transcription
of the various protein coding genes. So, what I told you in the summary, so far,
is, I gave a very brief overview of how actually the transcription initiation takes place in
e coli, and how the e coli RNA polymerase regulates gene expression, either in using
accessory proteins like activators and repressors in the case of operons, or by using specific
sigma factors, it can differentially regulate various bacterial genes. But, then I told, I had showed, I discussed
with you, as we moved from prokaryotes to eukaryotes the RNA polymerase itself became
very complex. So, instead of 1 RNA polymerase, you got 3
RNA polymerase in the case of eukaryotes, and in these 3 RNA polymerase, again, the
each of them became a multi-subunit complex, whereas eukaryote RNA polymerase has only
4 subunits and the sigma factor in the case of eukaryotic animals, you have anywhere from
9 to 12 subunits. Then, not only have different subunits of
the RNA polymerase, you also need a wide variety of accessory proteins in order for RNA polymerase
to go and bind to the promoter and recognize. And you can see, here, this is what is called
the… what I have shown here is what is called as a pre-initiation complex, which, actually,
is responsible for initiation of transcription in the eukaryotes. As I explained in my previous slides, you
have what is called as the TATA binding protein and you have what is called as the TF2B you
have the TF2A and what is called as a TBP associated factors, and these TBP associated
factors and TBP together constitutes what is called as a TF2B, TF2D. These TF2D, in addition to, and along with
TF2A and TF2B, are responsible for recognizing the core promoter sequence, namely, the TATA
box, in the case of the protein coding genes of the eukaryotes. Now, the job of TF2F is actually to target
the RNA polymerase to this DAB complex, which are actually formed, and once the RNA polymerase
2F complex comes and binds here, then the TF2H, which itself consists of a multi-subunit
complex, comes and joins the party, and TF2H actually has 2 major activities, namely, it
has a…, it is a helicase activity and the TF2H actually modulates the helicase activity. These factors then come and join the pre-initiation
complex, and when once all these entire complex is assembled in and around the TATA box, and
if ribonucleotides are present in the cell, and if ATP is present in the cell, then the
TF2H actually phosphorylates this C-terminal domain of RNA polymerase 2; and this phosphorylation
of CT domain is the actual signal that RNA polymerase can now leave the promoter, and
then go and start transcription of the RNA polymerase 2. So, you can see now how complex the whole
situation is; just to transcribe one gene, you require close to some 40 to 50 different
polypeptides, maybe 60 polypeptides. You require RNA polymerase 2, which itself
is a multi-subunit complex. You require TF2A, B, D, E, and F. Each of
them, again, is a multi-subunit protein complex and assembly of the all the subunits in and
around the TATA box results in the formation of what is called, say, pre-initiation complex,
and this pre-initiation complex is actually responsible for initiation of transcription
in eukaryotes. So, what I actually done in the next few slides
is actually give you some of the original research articles, starting way back from
1960 all the way down, and these are all very some of the keys experiments which are actually
done, to actually, to demonstrate how the eukaryotic RNA polymerase actually function,
how the purification of RNA polymerase were studied, and how the general transcription
factors or the key factors required for RNA polymerase 2 transcription were identified. These key original articles if you read, you
now, you get actually historical perspective of how difficult it was to study and understand
the transcription initiation process in eukaryotes, and it has not been a breezy road to some
of these studies, and lot of effort as actually gone into some of these things, and a number
of groups have actually contributed to understand transcription initiation eukaryotes. If you, some of these are all what I call
as a very important research articles, original research articles. They have actually laid foundation for our
basic understanding of eukaryotic transcription initiation. So, with this, I will now close this particular
lecture, and in the next subsequent lectures we are going to study little bit more details
about how actually is transcription initiation takes place; how each of the general transcription
factors function, and then, we will move on to other regions, the distal promoter elements
in the case of eukaryotes, and what happens there, and how other transcription factors,
transcription activators, interact with RNA polymerase 2. In the subsequent classes, I am going to tell
you, so far, I had discussed as if DNA is a naked template, but I am going to tell you
that DNA is not naked in eukaryote. It is actually present as a chromatin. So, all this RNA polymerase and general transcription
factors that we have discussed so far, they have to actually activate transcription not
from a naked DNA template, but they actually activate transcription from a chromatin template;
and how actually these transcription factors was able to recognize chromatin, and then
remove histones from the promoter regions, and then make RNA polymerase go and bind and
activate transcription in the eukaryotic promoters. This is what we are going to discuss the subsequent
classes. Thank you.


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