106 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
What is a gene?
A gene is one of the most talked about, but least
understood, topics in education. A gene is a length
of DNA that has all the DNA sequence the cell needs
to read and begin the Three Steps to Microfacturing. In
other words, a gene is a length of DNA that results in
the creation of an end-product that has a function,
like RNA or a protein.
This means a gene must have information embedded
in the DNA sequence to start and stop the Three Steps
to Microfacturing and to create a cellular product with
a function. In other words, a gene is a length of DNA
that can tell the cell machinery (RNA polymerase)
when and where to start the Three Steps to Microfactur-
ing as well as what to make. Let’s look deeper at how
it works.
Just like a sentence has a structure or “syntax”, such
as a subject-verb-object, genes have a grammati-
cal order. Two kinds of information are stored in a
gene’s DNA sequences. They are called “non-coding
sequences” and “coding sequences” (Figure 4-23).
These are both just plain old DNA. The RNA poly-
merase is able to distinguish between them.
A non-coding DNA sequence is a segment of DNA
that acts as a switch, controlling when and how much
product is made from the gene. The non-coding
sequence has the right characteristics to bind ‘tran-
scription machinery’, and it acts as the starting point
of transcription. Consider The Four B’s of Cell Opera-
tion: Bump, Bind, Burst, Bump. If the non-coding DNA
sequence is unable to bind the transcription machin-
ery, then transcription doesn’t happen. Conversely,
if the DNA sequence has the right shape and charge
to bond to the transcription machinery, RNA poly-
merase binds to the DNA more frequently, and
transcription can occur.
In the hands-on exercise, you engineered your cells
by adding a DNA plasmid that contains a gene for
creating protein color pigments. Within that gene is
a non-coding sequence designed to bind with a cell’s
transcription machinery ~12 hours after the cells
start growing and keep transcribing it thereafter.
A coding DNA sequence is a sequence situated imme-
diately next to the non-coding DNA. It is read and
transcribed by the transcription machinery into RNA.
The coding DNA sequences are like the designs for
the functional end-product that will be made from
the gene. The non-coding DNA sequences are like
the switch telling the cell where and how frequently
to transcribe the coding DNA sequence.
Starting Transcription
How does the cell know how to start transcription?
Both the DNA and transcription machinery are
bumping around the cell. If the non-coding DNA
sequence has the ‘right’ shape and chemical bonding
properties, it will bind to the transcription machinery,
enabling transcription to start. Let’s get more specic.
Small proteins called sigma factors complete the
Four B’s and eventually bind to short non-coding DNA
sequences within genes. For transcription, the small
non-coding regions are called promoters (Figure
4-23) because they “promote” the transcription of
the gene. Promoters are the starting points of tran-
scription, which means they are the starting point of
a gene. The sigma factor (σ) has a particular size and
shape that is able to bind to a specic DNA sequence.
Once a sigma factor binds to the promoter of a gene,
it then binds to the RNA polymerase. In other words,
the sigma factor acts as a bridge between the DNA
and the RNA polymerase, the enzyme that transcribes
RNA (4-23i).
Once RNA polymerase is bound to the promoter
region via the sigma factor, the RNA polymerase
creates a short RNA sequence called the initiation
sequence, which locks the RNA polymerase to the
DNA (4-23ii). The RNA polymerase then “drives off”
and escapes the promoter to begin unzipping, read-
ing, and transcribing the coding DNA sequence into
RNA (4-23iii).
An analogy can be drawn between transcription and
drag car racing (do a quick video search for ‘drag car
racing’). Note the similarity:
• Step 1: A race track ofcial walks onto the starting
line (a sigma factor binds to the promoter, which is
the ‘start line’ of the gene).
• Step 2: The ofcial waves the car forward and the
car advances to the starting line (the sigma factor
‘recruits’ and binds to RNA polymerase at the
promoter).
• Step 3: The car does a ‘burn out’, spinning its tires
to heat them up, making the tires nice and sticky,
so it has more traction (the RNA polymerase makes
a short RNA initiation sequence, locking into the
DNA).
• Step 4: Green light! The race car oors it and takes
off from the starting line (RNA polymerase leaves
the promoter and commences transcription).
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107Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Sometimes the car makes a perfect escape. However,
in some cases, the race car does a wheelie, burns
out, or the engine fails, and the car cannot effectively
escape the starting line. Often when RNA polymerase
attempts to start transcription, it cannot complete
all of these steps and fails to begin transcription.
This is called abortive initiation. If in the future you
want to design your own promoter DNA sequences,
keep in mind that controlling transcription is a ne
balance. You want the RNA polymerase to bind to
the promoter, but not too tightly or it won’t be able to
escape the promoter!
Transcription Video Breakout
You can search the web for some amazing videos that include computer graphic images of RNA polymerase
doing the Four B’s: Floating around, recognizing/binding to a non-coding DNA sequence, and then riding
along and unzipping the coding DNA sequence (reading it), all while transcribing an RNA molecule. Search
terms could include: “RNA polymerase video”, “DNA transcription video”.
Figure 4-23. The rst step of The Three Steps to Microfacturing is Transcription. Sigma factors recruit RNA polymerase to a DNA pro-
moter so that it can read DNA and transcribe RNA.
Non-coding DNA
“promoter” region
5’P
3’OH
Coding DNA
RNA
iii) Transcription ensues
RNAP
Non-coding DNA
“promoter” region
Coding DNA
DNADNA DNA
ii) RNA chain initiation
and promoter escape to
start transcription
RNAP
Non-coding DNA
“promoter” region
sigma factor
1
2
Coding DNA
i) Promoter binding
and activation
σ
RNA
Polymerase
(RNAP)
σ
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108 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Know your strand! Breakout Exercise
You’ve learned a lot of new information in the last few pages. Take a break to reect on what you know
about DNA by completing this short exercise.
Label the strand with the items below.
• 5’ Phosphate
• 3’ OH
• deoxyribophosphate backbone
• base pairs
• hydrogen bond
• hydrophilic
• ionic bond
Don’t forget! If you are stuck or want to verify your work, go to www.amino.bio/community
H
2
0
H
2
0
Ca
2+
H
2
0
3’OH
5’P
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109Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
During transcription: Direction
Just like roads have lanes that operate in different
directions, DNA and RNA have direction! Let’s get
oriented on how directions work in nucleic acids.
Recall that DNA and RNA nucleotides have a ribose
sugar that is bound to an OH group at carbon C3
and a phosphate group that is bound to carbon C5
(Figure 4-19). It is common language to say that the
phosphate attached at the C5 carbon position of the
ribose sugar is the “5’ phosphate” (‘ve-prime-phos-
phate’). The OH group on carbon C3 is referred to as
the “3’ OH” (‘three-prime-o-H’). The apostrophe is
pronounced as “prime”.
These two positions of the ribose are what become
connected to form a chain of nucleotides. If you look
again at Figure 1-17, you will see that at the beginning
of the strands there is a 5’ phosphate group that is not
attached to anything. Also, you’ll see at the other end
a 3’ OH that is not connected to anything. These two
end groups are how we understand the position and
directionality of DNA.
In the world of nucleic acids, the 5’ phosphate of a
DNA (Figure 1-17) or RNA (Figure 4-20) strand is
always considered the ‘beginning’. The 3’ OH of the
DNA or RNA is considered the ‘end’ and cellular
machines such as RNA polymerase, travel from the
5’ P toward the 3’ OH (Figure 4-20). A very common
way in which scientists describe the location in a DNA
sequence is using ‘upstream’ and ‘downstream’. The
5’ phosphate is upstream of the 3’ OH (Figure 4-24).
Genetic Engineering Heroes often use upstream and
downstream to describe a location or direction on a
strand of nucleic acid. For example, you can say “the
promoter is just upstream of the coding region”, or “I
get it, the coding sequence is just downstream of the
promoter!” (Figure 4-24).
Figure 4-24.
When using “upstream of” and “downstream
from” terminology, you refer to whether something is clos-
er to the 5’ phosphate (upstream) or the 3’OH (downstream)
of a specic strand. Be careful! You also have to know what
strand you’re referring to. Here, we are referring to the pink
DNA strand.
Upstream Downstream
5’P
3’OH
Point of Interest
Sigma Factors Going Deeper 4-8
Are there more than one type of sigma factor? Yes, there are many! The non-coding regions of genes have
co-evolved with many different sigma factors so that cells can have many different switches to turn genes
on and off. These enable the cells to create products at different times, under different growth conditions,
and within different environments! This enables cells to create certain products only when they are needed.
For example, a sigma factor called sigmaS (σS) binds to certain promoter sequences that control the tran-
scription of proteins that are important when cells are under starvation and are preparing to stop growing,
getting ready for ‘tough times’. When cells are growing fast, very little sigmaS is created by the cell. However,
as food becomes limited or the cells become crowded, the cells produce more sigmaS, which then can bind
to the non-coding promoters of genes and recruit RNA polymerase. This activates transcription resulting
in RNA that will later be translated into important proteins which ready the cell for ‘hibernation’!
Going Even Deeper
You may have just realized that sigmaS is a protein, meaning its creation through the Three Steps to Micro-
facturing must also be controlled by a sigma factor… and you’re right, it is! The coding DNA for sigmaS
is controlled by a promoter called rpoSp, which is also able to bind to another sigma factor. It gets more
complicated quite quickly, and it’s not necessary to dive deeper right now. However, if you’re interested,
you can do a web search on this topic.
In the context of the hands-on experiment you completed in this chapter, the gene for the color pigment
is controlled by a sigma factor that turns on when the cells enter a “stationary phase”.
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110 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
But DNA has two strands. What happens when they
come together? There are two directions, and each
depends on which strand you are referring to. When
two DNA strands zip together to form the DNA double
helix, the strands bind in an “antiparallel fashion”,
meaning that the 5’ phosphate ends of each strand
are at the opposite ends (Figure 4-25). Look back to
Figure 1-17 and notice where the 5’ phosphate and
3’ OH are located on the two strands. This means
that the ‘upstream’ and ‘downstream’ terminology
depends on which strand you’re referring to.
During transcription:
Which DNA
strand does RNA polymerase read?
There are lots of nucleic acid strands to keep track
of! During transcription there are three nucleotide
strands involved:
•
The RNA strand that is being made (transcribed)
by RNA polymerase
•
The two complementary DNA strands that make
up the DNA double helix, only one of which is being
“read” by RNA polymerase
How does RNA polymerase know which strand of DNA
to bind to so that it goes in the right direction?
The RNA polymerase binds to both strands simulta-
neously, but the direction it is pointing depends on
the strand that the promoter sequence is in. In Figure
4-27 you’ll see that the promoter (gray region) can
actually be situated on either strand, with the arrows
pointing in the downstream direction indicating the
direction that the RNA polymerase would travel. It is
a specic DNA sequence in the promoter which helps
lock the sigma factor and RNA polymerase in the right
direction (Figure 4-26). The DNA sequence ATCG’s
cause the DNA helix to have slightly different shapes
that the sigma factor can bind to in a specic orien-
tation. The orientation can help determine which
direction the polymerase points.
Whichever strand the promoter sequence is situated
in is called the ‘plus’ (+) strand or ‘leading strand’
and the RNA polymerase will be oriented so that it
will transcribe downstream of the promoter into the
coding region (Figure 4-26).
Here’s where things get a little wacky - ready for a
mind bender? Even though our point of reference is
the (+) strand, and the RNA polymerase travels from
5’ to 3’ according to the (+) strand, the RNA poly-
merase actually reads and transcribes from the other
strand, the template strand. Another name for this is
the ‘minus’ (-) strand. In the next section, we’re going
to see why RNA polymerase does this, along with the
‘cipher’ that it uses to transcribe DNA sequences into
RNA sequences.
Figure 4-25. Strands of nucleic acids have directionality,
meaning one end is different from the other. When two
strands of DNA bind together, they bind in an anti-parallel
manner so that the 5’ end of one strand is at the 3’ end of
the other.
5’ Phosphate
3’ OH
5’ Phosphate
3’ OH
5’
3’
3’
5’
Figure 4-27. The gray segment of each strand represents
different non-coding promoters. On the top strand, the RNA
polymerase would bind and continue to the right toward the
3’ OH end. On the bottom strand, the RNA polymerase would
bind to the gray region and transcribe to the left toward the 3’
OH end of that strand.
5’
3’ 5’
3’
+ strand for this gene
+ strand for this gene
Figure 4-26. RNA polymerase (RNAP) can bind to the DNA
nucleotides in one way which points the RNA polymerase in
the correct direction.
Promoter
Promoter
downstream
RNAP
downstream
RNAP
3’OH
5’P
3’OH
5’P
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