Eukaryotic gene transcription: Going

from DNA to mRNA

Genes are stored deep inside a cell, in a locked room called the nucleus.
Ribosomes, the machines that assemble proteins, live outside the nucleus,
floating around in a soup of chemicals called the cytosol. This spatial
separation presents a logistical hurdle for the cell. A ribosome needs the
instructions in a gene to put the corresponding protein together, but genes are
trapped inside the nucleus. How do the instructions in a gene get out of the
nucleus and to the ribosome?

The solution is simple (if you ignore the details). The instructions in a gene
(written in the language of DNA nucleotides) are transcribed into a portable
gene, called an mRNA transcript. These mRNA transcripts escape the
nucleus and travel to the ribosomes, where they deliver their protein
assembly instructions. The creation of mRNA transcripts (the creation of
these portable genes) is called gene transcription. Let’s learn about it.

An analogy for understanding transcription

Imagine that you are the owner of an Italian restaurant. You store all the
recipes that your cooks use in one big book and every night, when the kitchen
closes down, you store the book in your office for safe keeping. Now imagine
that one Saturday afternoon the door to your office has a major malfunction,
and you and the recipe book get locked inside. The restaurant opens for
business in a few hours. You try calling every locksmith in town, but nobody
is open on the weekend. How are you going to get the recipes from inside
your locked office to the cooks on the outside so that they can make the
dishes your customers order?

You come up with the following system. When a customer places an order
for a particular dish, you have the waiter come knock on your door and tell
you. You turn around and find the relevant recipe in the book, and then you
write it down on a 3x5 notecard. Because space is tight and time is of the
essence, you use some shorthand and abbreviations but make sure to include
all the essential elements of the recipe. You then put the notecard in a
sealable plastic bag (to protect it from damage in the kitchen) and slide it
under the crack at the bottom of the door. The waiter takes the card to the
cook waiting in the kitchen, and the cook has the information he needs to
make the customer’s dish. Problem solved. (If you ignore the fact that you're
still locked inside your office!)

The mechanics of transcription

In cells, transcription is the process that resembles copying a recipe onto a
3x5 card and sliding it under the office door. The 3x5 card, with the recipe
written on it, is analogous to a messenger RNA transcript (mRNA transcript,
for short). An mRNA transcript is a single strand of RNA that encapsulate the
information contained in a gene. Think of an mRNA transcript as a portable
gene: smaller and more mobile than the DNA sequence that it is built from,
but containing the same information.

What does an mRNA transcript look like?

 When learning about something new, it’s good to see if you can put it in
terms of something you already understand. In the case of mRNA transcripts,
the thing you already understand is a single strand of DNA (assuming you
have read our article on DNA structure and function).

If you hold a picture of such a DNA strand in your mind, you can turn it into
an mRNA transcript by making two changes.

 First, add a hydroxyl group to the 2’ carbon of each deoxyribose. In

biochemist speak, you need to hydroxylate the 2’ deoxyriboses.

 Second, snip the methyl group off of every thymine that occurs in the
nucleotide strand. In biochemist speak, you need to demethylate each
thymine.

Hydroxylated deoxyribose is called ribose. Demethylated thymine is called

uracil. A single strand of RNA is exactly like a single strand of DNA, in
terms of the chemicals it is made out of, except that it uses ribose in place of
deoxyribose and uracil in place of thymine.

It’s worth noting that cells don’t make mRNA transcripts by starting with a
single strand of DNA and then making the changes we just described.
Instead, they use a pre-existing supply of ribose and uracil, together with the
other components of nucleotides, to make mRNA from scratch. We are
simply suggesting that the best way to understand the chemical structure of
mRNA is to start with a strand of DNA and make the two changes described.

As another way of wrapping your head around the subtle differences between
DNA and RNA, have a look at the following chart.
 DNA RNA
bonds between
nucleotides phosphodiester phosphodiester

sugar in nucleotides deoxyribose ribose

adenine, thymine, adenine, uracil, guanine,

nucleobases guanine, cytosine cytosine

primary function information storage

How is an mRNA transcript made?

An mRNA transcript is made by an enzyme called RNA polymerase II. As
you can tell from the name, the function of RNA polymerase II is broadly
similar to DNA polymerase. The only high-level difference is in the building
blocks used.

DNA polymerase uses a single strand of DNA as a template and synthesizes a

strand of DNA. Each nucleotide in the synthesized DNA strand is
complementary to the nucleotide in the template strand. RNA polymerase II
also uses a strand of DNA as a template. Instead of using this template to
make a complementary strand of DNA, it uses it to make a complementary
strand of RNA — the mRNA transcript.

mRNA processing
Once RNA polymerase is done, the mRNA transcript has to be processed
before it can make its journey out of the nucleus and to the ribosome.
Processing has two phases: protection and splicing.
Protection
During this phase, nucleotide sequences are added to each end of the mRNA
transcript to protect it from degradation that can occur outside of the nucleus.
The 5’ end of a single G nucleotide is attached to the 5’ end of the transcript.
This is called the 5’ cap. At the 3’ end of the transcript, a long sequence of A
nucleotides are attached. This is called the poly-A tail. The 5’ cap and the
poly-A tail protect the mRNA transcript from attack by enzymes in the
cytoplasm called exonucleases that specifically target RNA molecules with
exposed 5’ ends.

Think of this protection phase of processing in terms of our restaurant

analogy. You know that you have to protect the 3x5 card with the recipe on it
from the damage that might occur to it in the kitchen, so you put the notecard
inside a plastic bag, in order to shield it from any water, oil, or other stray
ingredients that could compromise the integrity of the ink the message is
written in. The 5’ cap and poly-A tail have the same protective purpose.

Splicing
The other phase of mRNA processing is called splicing. The purpose of
splicing is to remove the introns from the mRNA transcript. Introns are
sequences of RNA that don’t contain any information about how to construct
a protein.

Introns are snipped out of an mRNA transcript by a complex of enzymes

called a spliceosome. A spliceosome locates introns, cuts them out, and then
fuses the remaining parts of the mRNA transcript back together. The parts of
the mRNA transcript that aren’t spliced out by the spliceosome are called
exons. In contrast to introns, exons are the part of an mRNA transcript that
actually contain assembly instructions for a protein. Many call the mRNA
transcript that still contains introns pre-mRNA, and the intron-free transcript
that the spliceosome produces primary mRNA (also called “mature mRNA”
by some authors).

Think of intron splicing in terms of our restaurant analogy. The recipes in the
big book may contain extraneous information, such as where the recipe came
from, its history in your family, or what other dishes or drinks it can be paired
with. For your purposes, this sort of information isn’t relevant to your
primary purpose. So you leave it out when you make the notecard, and only
include the need-to-know information for making the dish. In the case of
transcription, this need-to-know info is contained in the exons, and all the
rest—the introns—can be left out. The result is a smaller and more mobile
version of the mRNA transcript.

Once an mRNA has been protected and spliced, it is ready to leave the
nucleus and begin the second phase of protein synthesis, called translation.

Consider the following: RNA interference

In our restaurant example, what would happen if the notecard you slipped
under the door never made it to the kitchen? The dish wouldn’t get made,
because the cook wouldn’t have the recipe, right?

In recent years, a promising class of medical therapies have used a version of

this idea to develop new therapies for a number of formidable diseases.
Falling under the name of RNA interference, these therapies disrupt the
production of harmful proteins by intercepting and incapacitating mRNA
transcripts before they make it to ribosomes. This prevents the corresponding
proteins from being made in the first place!
A recently proposed treatment for Ebola represents perhaps the most
spectacular application of RNA interference techniques.

Ebola, like most viruses, is basically a transcription machine. It contains a

viral genome --- basically a very small and simple chromosome --- together
with a polymerase enzyme. Once the virus infiltrates one of your cells, the
polymerase enzyme synthesizes mRNA transcripts for each of the genes in its
genome. It then commandeers your own ribosomes and uses them to build its
own proteins. Once you make these proteins for the ebola virus, they direct
the construction of new ebola viruses.

One obvious idea for fighting ebola would be to throw a monkey wrench into
this whole process, and stop the replication process before it ever gets off the
ground.

Using a class of engineered molecules called short interfering RNA (siRNA,

for short), scientists have shown that the viral mRNAs synthesized by the
Ebola virus inside infected cells can be captured and destroyed before they
are able to deliver their genetic messages to the ribosomes of a host cell. No
Ebola mRNA, no Ebola proteins. No Ebola proteins, and Ebola loses its
ability to replicate inside host cells!

As with all other new therapies, siRNA-based treatments for Ebola were
initially validated in non-human animal models. However, during the latest
outbreak of the virus in West Africa, and its subsequent spread to North
America, authorities in the U.S. Food and Drug Administration took the
radical step of issuing a so-called “compassionate use” exemption for an
siRNA-based Ebola therapy called TKM-ebola. While the details are sketchy,
we know that TKM-ebola was administered to several different patients, and
it could have played a role in their subsequent recoveries.