OK. Parts of a gene.
We have our promoter, which is part of the untranscribed
region of a gene, usually in the 5 prime end.
Not always but for the genes we're talking about at the 5 prime end,
the so-called 5 prime end of the gene, or so-called upstream of this
transcribed region. And downstream of that there is more
untranscribed region that interestingly can also contribute to
the promoter, even though it's far away from this more upstream part of
the promoter. But I'm going to call it just for now untranscribed,
two flanking regions of untranscribed DNA sequence and one
region of transcribed sequence. Now, I want to discuss with you very
briefly a phenomenon called splicing. And this is a phenomenon that
occurs within the RNA that is transcribed from a gene and,
therefore, pertains to the transcribed region of the gene.
It turns out that in this transcribed region there are two
kinds of sequences. There are things called exons and
there are regions called introns. The exons code for something,
code for the final function of the RNA or for eventually a protein.
So these are coding. The introns are noncoding.
Both of them are transcribed. You'll see this definition is a
little loose as we move on in today's lecture, but
it's good enough. In the transcript that initially is
made from the gene in this transcribed region,
both introns and exons are present. So these are present in what's
called the primary transcript or primary RNA. And primary refers to
the first RNA that is transcribed from the gene.
And subsequent to that, still in the nucleus, those introns
and exons are subject to a process called splicing whereby the introns
are removed --
-- or spliced out is the term, such that in your mature RNA only
the exons are present.
This process is likely a consequence. I'm going to put up a diagram that
you had on your last time's handout. You can watch it now. And you can
refer back to a previous lecture if you don't have it with you.
This notion of introns and exons is probably a consequence of evolution
whereby different parts of genes were combined and shuffled to give
new kinds of genes and, therefore, new kinds of proteins.
Here on my diagram I have exons in black and introns in blue,
and they're all just DNA sequence, but when the RNA is transcribed in
the first place is primary RNA. It's a copy of the gene.
It has both exons and introns. And then a very complex enzymatic
machinery comes on and it loops out and excises these introns.
OK? So this is very interesting such that in your mature mRNA there
are no introns. And the introns have been looped
out and they form these little structures that are called lariats.
And at this point your mRNA is mature --
-- and it moves to the cytoplasm. Now, this process what discovered
by Professor Phillip Sharp here at MIT and he got the Nobel Prize for
it in 1993. It's a very important process because it's absolutely
required for maturation of RNAs. And also, and I'll come to this in
a few lecture's time, it allows different proteins to be
made from the same mRNA. So here's a rule.
In this RNA there are what are called splice donor cites that I've
put as a circle and splice acceptor sites that I've put as a square.
Just watch this. Just watch this for now because we will come back to
it. So watch what I'm saying rather than trying to madly write down.
Any spliced donor can join to any splice acceptor and remove the stuff
between them. So in this top example I've got each introns being
neatly removed because splice donors and inceptors interact.
But look at the example below. I've got this splice donor next to
exon one interacting with a spliced acceptor next to exon three.
And when that happens you remove the hull of exon two.
So you actually are going to make a different protein.
Whereas, in the first case you'll have exons one,
two and three and four. In the second case you'll have
exons one, three and four. OK? So this process is very
important for allowing different kinds of proteins to be made from
the same gene. I want to make you aware of this
now, and I will come back to it in the formation module when we talk
about how different kinds of cells are generated.
All right. So let's move onto the major topic of today's lecture --
-- which takes us back to the central dogma.
And I want to introduce to you a term that is very important that you
know and you understand. And this is the term gene
expression. And really what we've been talking about is
gene expression.
Gene expression simply refers to the generation of the final product of a
gene from the gene. So we're talking about the
formation of a protein as directed by a particular gene.
OK? So gene expression is, if you like, the readout. Here's
another way of putting it. The readout, the final readout of a
gene, or the generation of the final product of a gene.
I'm going to come back to this term over and over again,
and I will ask you to define it in your own way, but it's a term I want
to throw out at you now because you do need to know it.
It's very pervasive. Today I want to talk about the step
in gene expression or translation whereby RNA is converted or is used
to direct synthesis of a protein. So let's define translation because
it is, I think, one of the most interesting
questions in molecular biology. Certainly from a historical
perspective that was true. And the notion in translation is
that the base sequence of a mRNA somehow leads to the synthesis of a
protein with a defined amino acid sequence.
Now, if you think about DNA
replication, transcription and translation, the relationship
between them, there is a nice analogy that one can make.
DNA uses the base code, four bases. Transcription RNA uses those same
four bases as a code, but it's slightly different from DNA.
So the synthesis of RNA using a DNA template is kind of like changing
fonts in a document that you have. It's kind of like going from Times
New Roman to Helvetica. You haven't really changed much.
It just looks a bit different. Translation is very different.
The use of mRNA to direct the synthesis of a protein is much more
analogous to changing language where you've taken English and translated
it into Chinese or Russian and translated it into French.
OK? So this is a really different process. And it was clear from the
outset, historically, that one had to think in a slightly
different way about how this process was directed.
And I want to talk about four things with respect to translation.
Firstly, I want to talk about the genetic code that allows RNA to
direct protein synthesis. I want to talk about something
called the interpreter of that code.
I'm going to talk about the factory in which the synthesis takes place.
And then I'm going to get to a discussion of the molecule bases for
genotype and phenotype.
So let's think about the code. And thinking about this starts from
a very simple logical place. And the place is this. One starts
with four bases, A, G, C and T or A,
G, C and U, depending if you're talking about DNA and RNA.
And somehow those four bases have to be used in some kind of code to
give you an outcome of 20 amino acids.
And I am going to use the abbreviation AA for amino acids.
So you can look at this and immediately understand there has to
be some kind of combinatorial code in order to specify those 20 amino
acids. So you can do combinations and you can say,
OK, if two bases were used and you could have combinations of doublets,
how many combinations can you get to and would that be enough to specify
those 20 amino acids? Well, no, because two base
combinations would only give you 16 possible amino acid combinations,
or the ability to specify 16 amino acids. OK? Four squared.
How about three base combinations? Well, that's better.
What you can get out of that is 64 different combinations.
OK? And that is plenty to specify your 20 amino acids with some left
over. And, in fact, this is what is used.
Combinations of three bases. And these combinations of three
bases are termed the triplet code.
The discovery of the triplet code is really fascinating.
I don't have time to go into it in this lecture, but your book is not
too bad on the discovery. And I will post on your website,
for those of you who really want to get into it, a reference to a very
interesting historical account of the discovery of the triplet code
and indeed of much of molecular biology. But it's a fascinating
story. But I'm going to tell you the code is a triplet code. OK.
So what does that mean? It means that three bases
correspond to a particular amino acid. OK? So one triplet of bases
correspond, I'm writing this out because it's really important that
you know this, correspond to one amino acid.
And this base triplet gets a special name. It's called a codon.
And the thing that you will have
noticed is that what I've told you is there are 64 possible
combinations of triplets and only 20 amino acids. And so that leaves
some over. What happens? Well, they're all used.
And what happens is that although the code is universal,
as far as we know it arose just once, all living organisms on our planet
use this code, it is a redundant code.
So I will write down it is redundant but not ambiguous,
and tell you what that means. So what that means is that an amino
acid can be specified by more than one triplet, and I'll show you that
in a moment, but that any triplet of bases only corresponds to one amino
acid. Let's look at some diagrams to show you what I mean.
This is a table of your amino acid code.
These letters in columns represent the bases. And next to them are
written the amino acids that correspond to this particular code.
Let's start with an easy one. This is methionine encoded by AUG.
And that's one you should actually remember. OK?
And for methionine there is only one possible codon.
It is AUG and always AUG. But let's keep going here.
And let's look at the amino acid lucine.
lucine is encoded by six possible triplets, six possible codons,
UAA, UAG, CUU, CUC, CUA and CUG. Any one of those in a mRNA can
encode lucine. However, CUU only encodes lucine.
It never encodes another amino acid. OK? And that's what
I mean by redundant. More than one triplet can encode one
amino acid, but any given triplet only corresponds to one particular
amino acid. OK. You will have practice on this kind
of thing as you go along. So let's get some basics down here.
The template in the whole translation process is your mRNA.
OK? It's the code. It contains the code.
It is read to give a protein readout from 5 prime to 3 prime.
And the readout of the protein, as I mentioned to you way back when,
reads out from the amino to the carboxyl end. New amino acids are
added onto the carboxyl end -- -- and the free amino group
corresponds to the first amino acid polymerized. So it is read 5 prime
to 3 prime, and that corresponds to the amino to the carboxy
growth of the protein.
All mRNAs start with the same amino acid, and that is methionine.
And the start or initiation codon in all proteins is methionine,
oops, is AUG which encodes methionine.
Now, not all final proteins have got methionine at their amino ends
because it can be cleaved off. OK? So you don't have to land up
with a protein that has a methionine end, its amino end,
but it starts off with methionine there. And then there are no gaps
in the message. It is read without any punctuation
marks, except for the fact that the codons are next to one another in a
non-overlapping way. OK? So there are no gaps.
And the only punctuation is the start codon and a series of stop
codons which do not encode any amino acids. These are UAA,
UAG and UGA. And you can remember them if you want,
but we're not going to test that you do. OK? You can use your
amino acid tables. OK. So your punctuation is the
start and the end of the message. All right. So let's go on and talk
about the interpreter and what I mean by the interpreter.
In this diagram here I have got, look up here for a moment. This is
quite a nice diagram not from your book. I've got your DNA strand,
which is your template strand. Your corresponding RNA,
your mRNA, and the readout of the RNA to the protein.
And here are the codons, UGG, this is in the middle of the
protein so that's why there's no methionine, UGG corresponding to
tryptophan, UUU corresponding to phenylalanine.
You can see how the codons are right next to each other,
OK, but do not overlap. In fact, I'm going to write that on the board.
So no gaps and no codon overlap. Very important that you understand
that. So when people looked to this and figured out what the codons
corresponded to in terms of amino acids there was the question of,
well, how do you actually get those amino acids corresponding to those
codons? And there was a sense that you needed some kind of adapter or
interpreter molecule that both recognized the codon and recognized
the amino acid. And that's the next thing that I'm
going to tell you. And --
-- stop. Well, I apologize on behalf of our
illustrious institute for the boards in this room. OK.
So all right. So let's talk about interpreter.
And I'll tell you that this is the class of RNA someone brought up
earlier called tRNAs. So tRNA, as you may recall,
are these very small RNAs. There are about 100 base pairs,
100 bases in length, and there are a lot of them. And there is a tRNA
that corresponds to every codon. So tRNAs recognize both the amino
acid and the specific codon. And they recognize,
let's talk about the codon first. They recognize the codon by DNA
complement, by RNA complementarity, by base pairing to a region on the
tRNA called the anti-codon.
So let's talk about methionine for a moment. The codon for methionine is
AUG. That's the codon. Woops. Hold on one second here.
5 prime AUG, that's your codon. And what will be complementary to
that on the tRNA from the 3 prime end is UAC.
OK? So this anti-codon is on the
tRNA. Anti-codons can either be written from the 3 prime end or you
can switch them around and talk about 5 prime CAU. It's
the same thing. OK? So that's one thing.
I'll show you a picture in a moment. The other thing that a tRNA has to
recognize is the amino acid. And that's more complicated.
For different amino acids there are different parts of the tRNA molecule
that recognizes specific amino acids. And it hasn't actually been figured
out completely which part of which tRNA recognizes a particular amino
acid, but the recognition is also on the tRNA --
-- and not really on the anti-codon. Or certainly not the anti-codon
alone is probably fair to say. So let me show you a picture of a
tRNA. tRNAs are single-stranded RNAs that fold up on themselves in a
complex way. OK? Here's the representation of the
three-dimensional structure of a tRNA. And these cross things
are hydrogen bonds. So there's a lot of base-pairing
within the tRNA. Represented more simply,
the tRNA forms this kind of cloverleaf structure,
and the anti-codon is at one end of the tRNA. OK?
So this is the thing that's base pairing to the codon and the mRNA.
The amino acid attaches to the very 3 prime end of the tRNA at this site
which is a CCA. OK? And there is a covalent attachment
of the tRNA to the amino acid at this CCA region.
All right. But the part that recognizes the amino acid can be
somewhere in the rest of the tRNA molecule. It's very complex.
OK. So let's move on now. Actually, let me tell you one more
thing, though I'll tell it to you in a moment. OK.
So let's move on now to the question of the factory.
And by factory I mean the place where protein synthesis or
translation takes place. And the factory here is the
ribosome. We mentioned ribosomes right at the
beginning of the course in the second lecture and haven't said a
whole bunch about them since. Ribosomes are very large structures.
They are not membrane bound, but they are very large. This is a
representation of a ribosome from bacteria that has a small subunit
and a large subunit. And, interestingly,
ribosomes are an obligatory complex between the so-called rRNA,
or ribosomal RNA, plus proteins. There is a small subunit, this is
really bad. Let's try this one. Small subunit which consists of one
ribosomal RNA of a particular kind and 33 proteins.
And there is a large subunit. And I tell you this not because you
need to remember this, but you need to appreciate that this
is a very complex structure. It's a very cool and complex
structure. The large subunit comprises of three RNAs
and 45 proteins. You can represent the structure of
the ribosome much more beautifully in this diagram,
or in this representation, where the RNA is shown in gold,
or the two RNAs are shown in gold, or the multiple RNAs are shown in
gold, and some of the proteins are shown as these other structures and
you can see the alpha helices of the proteins. OK?
And what you should be able to see on this diagram,
let me point to this one for a change, is this tunnel,
this hole through the structure. And this is the tunnel through which
the mRNAs thread as it is translated. So this is truly a factory.
tRNAs come into this, the mRNA threads through,
and as that takes place so the mRNA directs the synthesis of the protein.
OK. This is a representation from your book. I don't like most of the
diagrams from your book so I redrew most of them for you,
but I left this one. This is a representation of translation.
The mRNA is shown in green and the large subunit and small subunit of
the ribosome come together, form the complete ribosome, and then
the mRNA actually is thread through the ribosome and the protein,
well, here they've called it a polypeptide chain is thread through.
So let's explore this in a big more detail. And in order to do so,
I've got to conserve boards here because we are one board short.
In order to do so I need to introduce you to the various parts
of a mRNA. And this is on one of the diagrams that I handed out today.
OK? So you don't need to redraw it. Just look at the diagram.
In the mRNA, and this is crucial for translation,
there are three parts that are really important. Two
of them, excuse me. Two of them are actually added to
the mRNA after it is transcribed. The thing at the very 5 prime end
called the cap and something at the very 3 prime end,
which is a long string of up to a couple of hundred A residues
contiguous, which is called the poly A tail. And these parts of the mRNA
are crucial for the first part of translation which is initiation.
As in replication and transcription, you can divide up these synthetic
processes into different steps. And initiation is the first step.
And in order for initiation to occur one needs the parts of the RNA
that are added on post-transcriptionally.
You need the cap, this poly A tail -- -- and also a region that is just
upstream or 5 prime of this AUG initiated codon in a region called
the UTR, the 5 prime UTR which stands for untranslated region.
And you also need the AUG codon. OK. And what happens is that the
ribosome and various initiation proteins bind to the 5 prime cap and
simultaneously to the poly A tail. So this is really cool.
The mRNA is translated as a circle where this poly A tail,
the very 3 prime end is brought all the way around to the 5 prime end,
and you get a whole mess of proteins sitting on that part of the RNA and
starting translation. So you get initiation proteins,
which are called initiation factors, and you get ribosome assembly where
the small subunit and the large subunit come together,
and you get a tRNA carrying a methionine amino acid coming and
sitting on the AUG.
OK. Let me show you more. So here we have a cartoon,
you have this in front of you but I'm going to show it to you in a
step-wise fashion, of this ribosome recognition
sequence. Actually, I'm not going to show you now but in
your handout there are pictures of the circular RNAs being translated.
OK? That's something new and it's something very interesting.
I'm not going to dwell on it now. OK? Where the poly A tail comes
all the way around to that 5 prime so-called cap region.
I should just point out, again, I'm not going to dwell on it,
the so-called 5 prime cap region is a modified guanine.
OK? MEG stands for methyl guanine. You can call it the cap. It
designated the very 5 prime end of the message.
OK. So let us look at the sequence of translation.
And what I'm going to tell you, before I go through the cartoon, is
that in the elongation process sequential tRNAs carrying their
particular amino acids are going to come in.
And they're going to sit on these various codons.
And peptide bonds are going to form between adjacent amino acids so you
get the polypeptide chain growing. OK? So let's start off with the
initiator. There's your tRNA that is joined to methionine.
And I need to introduce you to a term now which is a charged,
I didn't have space before. The term charged tRNA refers to the
tRNA covalently linked to its amino acid. And then correspondingly the
uncharged tRNA has no amino acid. The amino acid has fallen off or
been used. OK. So there is a tRNA sitting on the
first codon, the AUG, and that's the start of the sentence.
That positions the beginning of the protein. Now,
watch what happens. Here comes another tRNA that
corresponds to lucine, and you're getting base pairing here.
That first tRNA is base paired to the AUG codon through its anti-codon.
The second tRNA is base paired to the second codon through
its anti-codon. And now you've got a methionine tRNA
sitting next to a lucine tRNA. OK. Everyone with me here? And
what happens now is that a peptide bond forms between the methionine
and the lucine. In particular, this methionine is
going to move over to that lucine over there and lead to uncharging of
that particular tRNA. Take a look. OK,
so I've shown you that methionine is going to form a peptide bond with
the lucine. Now, watch what happens next.
Here's the methionine tRNA. It's lost its amino acid, OK,
so it falls off the message. It's done its thing. It's
no longer needed. Along comes, no,
sitting there is this lucine tRNA which is now covalently attached to
its peptide bond to the methionine. And there's a free amino end here
which designates the first amino acid synthesized in a polypeptide
chain. And here comes in the next tRNA that corresponds to a serine
tRNA based paired by its codon, base paired by its anti-codon to the
codon on the mRNA. OK? And the same thing is going to
happen again. The lucine and the methionine is going to be
transferred over and make a peptide bond with the serine,
and so you get elongation of the polypeptide chain.
So what I'm going to write under elongation is that adjacent
amino acids join.
Uncharged tRNAs leave, are released, and sequentially new
tRNAs corresponding to codons come in.
OK. All right.
So this whole process goes on until the mRNA, until the ribosome and all
these tRNAs reach a place in the mRNA where there is a codon that
doesn't correspond to an amino acid.
A so-called stop codon. And at this point there is a
process called termination where there is a stop codon that does not
code for any amino acid and doesn't have a corresponding tRNA therefore.
And at this point the protein polypeptide chain falls
off the message.
All right. You guys OK with that? OK. I'm going to refer you, I'm
not going to go and watch this movie. Go and watch this movie.
Go and watch the movie by yourselves. OK?
I don't want to take the time to watch it now. It's an animation of
what I've just told you. There are some diagrams in your book.
You can look at them. They talk about things called A
sites and P sites in the ribosome. To me that is less important than
you understand the actual interactions between the tRNAs and
the mRNAs. Here is a circular RNA with that poly A tail and the 5
prime cap of binding proteins to initiate translation
as a circular RNA. All right. So,
finally, let's move to this complicated, I think fantastic
bringing together of mutation from genotype to phenotype.
You've had a genetics module where you talked about mutations,
you talked about the genotype, you talked about the phenotype.
We've been throwing at you genotype has got something to do with the DNA
base sequence. Phenotype has got something to do
with the final product, particularly the protein sequence.
Let's explore that in a bit more detail now and ask,
what is the molecular basis for changes in genotype and how do these
correspond to changes in phenotype? OK. So genotype to phenotype.
And I want to emphasize again that phenotype is an outcome of a change
in function of the final product of a gene. It isn't necessarily the
same as the final product of a gene. OK? So, for example, a phenotype
is giantism. Someone who is very tall. The molecular basis for that
could be multiple things. It could be production of too much
of a hormone, a protein called growth hormone so that someone grows
too tall or taller than normal. OK? So that is the phenotype is
connected to the production of a particular protein that's not the
same as. So here's another diagram, something for you to think about.
Mutations, almost anywhere in a gene, can have an affect on the protein
produced. And there are two ways the protein produced
can be affected. One is in the amount of protein and
the other is in the sequence of the protein produced.
Now, if one gets a mutation in this promoter region or often in the
introns, but particularly the promoter I've focused on,
one can change the amount of RNA that is being transcribed from a
particular gene. And that change in the amount of
RNA will lead to a change in the amount of protein.
And you may get a phenotype because you're making too little or too much
protein. Conversely, changes in exons can lead to changes
in the actual sequence, the amino acid sequence of the
protein and, therefore, to its function. So those are two
important distinctions to make. OK? So mutations can change the
amount or the sequence of a protein.
I'm going to go through some examples of mutations,
and you will go through more in Section, and you are expected to
know these changes and you are expected to know how the change in
DNA sequence may lead or not to the change in protein sequence.
So look carefully. I'm not going to get through all my
examples today. You can go and you can do the
examples that are posted on your website. You'll get more practice.
And you really need to know this. OK, so here's a wild type gene.
The top two strands are the DNA. The bottom of the strands is the
template strand. This DNA is transcribed into a mRNA
and that is translated into the protein indicated here.
OK? Let's look at an example of what happens when there is a change
in the DNA. So here I've got a change in the DNA,
OK, such that this particular base pair has been changed.
The mRNA, oh, this is the wild type again. Here's your wild type
sequence, wild type mRNA, wild type protein.
This is a class of change in the DNA that is called a nonsense mutation.
Watch carefully. So at this position that I've underlined,
watch this. Don't try to write anything down.
OK? You'll have plenty of practice. This is all posted.
Just watch. At this particular underlying position,
instead of a GC base pair there is now an AT base pair.
And that changes this codon UGG into UAG. And UAG happens to be a stop
codon. So here's your gene, your mutant gene, here's your mRNA
that comes from the mutant gene, and here's the protein. It starts
OK with a methionine. But, look, the next codon is a stop.
OK? So the protein is truncated. Now, there are a number of classes
of mutation. I am going to write these on the board.
I'm going to ask you to go and read your handout carefully.
And you will cover these in section. Again, you need to know them so let
me list the types of mutation. In the interest of time, I'm not
going to go through them, but you will be able to work through
these examples both in Section and on your own.
So, to end off, the mutations in exons that you
should know are silent mutations that don't change the sequence of
the protein, nonsense mutations that I've just covered,
something called missense mutations which change the amino acid sequence,
and something called frameshift mutations which also are likely to
change the sequence of the amino acid.
OK? As I say, this will be covered.
If you want to come and see me personally in office hours tomorrow
or the next day, please do, and I'll go through these
examples with you.