BIO230 lecture 4 notes
- Transcription is conducted by RNA polymerase that transcribes DNA into RNA.
- There are different types of RNAs that can be produced by RNA polymerase; we will be talking
about mRNA because it is the one that produces proteins. Recall that eukaryotes have 3 types of
RNA polymerase and prokaryotes only have one; we talk about RNA polymerase II because it
- In eukaryotes, the initiation of transcription requires 5 general transcription factors. Recall that
in prokaryotes, the RNA polymerase only needed sigma factor. These transcription factors help
position the RNA polymerase at eukaryotic promoters and these promoters have a specific
sequence called the TATA box that is located 25 nucleotides upstream of the transcription
initiation start site; it is called TATA because of the nucleotide sequence found there.
- These general transcription factors are required for all promoters that are used by RNA
polymerase II. TFIID recognizes the TATA box; this then recruits TFIIB, which then tells RNA
polymerase II where to start. TFIIH is recruited along with RNA polymerase II and it is involved in
unwinding the DNA and in phosphorylating the C-terminal tail of RNA polymerase; this
phosphorylation kick-starts transcription. Once all of these transcription factors get onto the
promoter, then transcription can start.
- Eukaryotic genomes do not have operons (some exceptions); this means that every gene has its
own RNA molecule and promoter. They are also packaged into chromatin, which provides an
additional mode of regulation. The added complexity allows genes to be differentially regulated
much more readily in eukaryotes relative to prokaryotes.
- There are many gene regulatory proteins in eukaryotes, with a main one being the mediator.
The mediator acts as an intermediate between regulatory proteins and RNA polymerase.
It provides a larger surface area than RNA polymerase such that many more gene regulatory
proteins can be interacted with.
- In humans there are about 2000 gene regulatory proteins, including activators and repressors.
One feature of eukaryotic gene expression is that these regulatory proteins can act over very
large distances (over 10 000 base pairs away); the mechanism that allows this to happen is DNA
looping (similar to lac operon).
- Gene regulatory proteins are usually found as protein complexes on the DNA. The complexes
bind to the DNA and are complex to an additional one; this makes them coactivators because it
contributes to activation of gene expression. This is similar to repressors and they are called
corepressors. Coactivators and corepressors assemble on DNA-bound gene regulatory proteins
but they do not directly bind DNA themselves. Refer to slide 36 (or 6 slide in lecture 4) for
- A key feature of eukaryotic activator proteins is that they have a modular design; this means
that they have 2 modules that carry out 2 distinct functions.
One of those modules is a DNA binding domain; this domain is responsible in recognizing
specific DNA sequences and binds to DNA.
The second module is the activation domain and it accelerates the rate of transcription.
These modular domains are separable and interchangeable with other activator proteins.
Refer to slide 37 (or 7 slide in lecture 4):
The Gal4 and the LexA transcription factors are both modular; they have an
activation domain and a DNA binding domain. The Gal4 transcription factor activates a gene called galactokinase; it has a DNA
binding domain that binds to the green cis element in the promoter of the
galactokinase gene and it has an activation domain that accelerates the rate of
You can take away the DNA binding domain and swap it for the LexA one, thereby
making a chimeric transcription factor.
Since the binding domain has been swapped, it doesn’t activate the expression of
But if you put the LexA cis element in the promoter, it can transactivate the
expression of this gene because now it has the LexA DNA binding domain and it can
bind to the LexA cis element, and use the Gal4 activation domain to activate
- How do activator proteins activate transcription in eukaryotic cells? Remember that their overall
goal is to do one of three things: attract, position, and/or modify either the general transcription
factors, mediator proteins, or RNA polymerase. They can do this by either directly acting on
these concepts or indirectly modifying chromatin structure; this is the same for activator
proteins in prokaryotes (but it wouldn’t alter chromatin structure, it would alter DNA structure).
The direct mechanism: The activator protein binds directly to the transcriptional machinery
or mediator and attract them to promoters; this is just like prokaryotic activators.
The indirect mechanism:
Recap that eukaryotic DNA is packaged into chromatin and if you were to extend DNA, it
would look like “beads on a string”.
These “beads” are nucleosomes that occur about every 200 nucleotides. DNA is
wrapped around a histone octamer, with each octamer being made up of 4 proteins:
H2A, H2B, H3, and H4 that are repeated twice.
There is linker DNA between each histone molecule.
There is an additional structure imposed on the nucleosomes where they are packaged
into chromatin fibres: they wrap around each other to form this very compact fibres.
The structure of these fibres is controversial:
One crystal structure made with a tetra nucleosome looked like a zig zag model
Another technique using a much longer piece of DNA looked like the solenoid
The overall result is that the transcriptional machinery cannot assemble on promoters
that are tightly packaged into these chromatin fibres.
- Activator proteins alter chromatin structure and increase promoter accessibility. There are 4
ways to do this, 3 of which involve the recruitment of chromatin remodelling complex.
Gene activator proteins recruit histone-modifying enzymes. These enzymes create a
specific pattern of histone modifications in an area of the DNA and this serves as a code
for signalling chromatin remodelling and thereby, gene transcription. This code/pattern
is called the histone code.
What types of modifications do these enzymes do?
They can add phosphate groups to histones via a process called phosphorylation
by using a kinase enzyme. The second modification is the addition of an acetyl group through a process
called acetylation that is carried out by acetyl transferases.
The third type of modification is the addition of a methyl group through a
process called methylation using methyl transferase/methylases (same name,
All of these modifications occur on histones at specific amino acids at specific
regions of the histone molecules; these regions are called histone tails. Extending
out from the nucleosomes are these tails and they stick out of the compact
structure, making themselves amenable to these modifications.
These proteins that write the histone code induce specific modifications to histone
tails and these proteins are called writers; they are the histone modifying proteins:
kinases, methyl transferases, and acetyl transferases. Each decoration of
modifications has a different meaning. The complexes that are involved in decoding
these meanings are called readers; they are the code breakers that recognize
specific modifications and give meaning to the code.
Slide 48 (or 18 slide of lecture 4) shows modifications made on histone H3 at
specific amino acids. When read by reader proteins, they contribute different
information in terms of gene expression.
Example: how the human interferon gene promoter is act