111Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
How does RNA Polymerase know which direction to go? Going Deeper 4-9
You just learned that DNA is non-symmetrical and has “directionality”. This means that the ends of the DNA
strands are different. You also learned that RNA polymerase travels in one direction, from 5’ phosphate
downstream to the 3’ OH. So how does the RNA polymerase know which way to go? A simple answer is that
RNA polymerase has evolved to go in one direction. Similar to how gears can be designed to turn in one
direction (see: https://amino.bio/pages/ratchet-gear ), RNA polymerase operates only in the 5’ phosphate
to 3’ OH direction.
But how does the RNA polymerase point toward the 3’ OH so it does travel in the right direction? Figure 4-26
shows how some nucleotides can interact with the surface of the RNA polymerase and cause it to point in
a direction. While this illustration is a simplied version of what happens in real life, you’ll see that there
is only one orientation in which the RNA polymerase can bind, and this will point the RNA polymerase in
the correct direction.
Bidirectional transcription in a plasmid? Going Deeper 4-10
Have a look at Figure 4-28. This is an adapted illustration of Figure 4-13 where the twist of the DNA helix
was removed to show each DNA strand more clearly.
•
Your trait/gene: is designed so the RNA polymerase will bind to the promoter (5’P) and transcribe
towards the 3’OH direction of the pink (outer) strand. While this happens, it uses the red (inner) strand
as a template to create an RNA molecule version of the pink strand.
•
Selection gene: is designed so the RNA polymerase will bind to the promoter (5’P) and transcribe
towards the 3’OH direction of the red (inner) strand. While this happens, it uses the pink (outer) strand
as a template to create an RNA molecule version of the red strand.
The Ori is not involved in transcription. This bidirectional design is often used so that the RNA polymerase
from one gene does not continue into the next gene and transcribe it as well. This could decrease your
control of the genetic system!
In the gure on the left, even if the RNA polymerase continues past your trait gene, it will not transcribe the
selection gene because that RNA polymerase is reading the wrong strand. On the right it is possible that
transcription starting in a gene can run through another gene!
Figure 4-28. Left: Bidirectional transcription in a plasmid. Right: Unidirectional transcription in a plasmid.
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Long RNA molecule that can
be read by the ribosome!
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112 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
During transcription: A secret
cipher for transcribing DNA to RNA
After discovering DNA’s structured in the 1950s, the
next great mystery was how DNA could hold the infor-
mation for other molecules such as RNA and proteins.
It took decades of research to unravel this.
As you saw in Figure 4-19 and Figure 4-20, RNA nucle-
otide structures are quite similar to DNA nucleotides.
There is one other significant difference between
DNA and RNA: There is no ‘T’ thymidine nucleotide
in RNA. Instead, there is a ‘U’ nucleotide for uridine,
hence, GCAU.
In Chapter 1, you saw that certain nucleotides can
complement each other using Chargaff’s Rule. Nota-
bly, A’s can bind to T’s to form a double-stranded DNA,
and C’s can bind to G’s. This complementary rule is
how RNA polymerase knows which ribonucleotides
to match up with when transcribing an RNA strand,
and this is why it always reads the template strand. It
uses the template strand because it is a ‘mirror image’
of the leading (+) strand. The end goal of RNA poly-
merase is to have an RNA strand that is a replica of
the leading (+) DNA sequence, making the (-) template
strand its ‘mirrored strand’.
Lastly, because there is no ‘T’ in RNA, T’s are replaced
with U’s, (Table 4-2). This means that the ‘A’ in a (-)
template strand interacts with a ‘U’ instead of a ‘T’.
In other words, if there is a ‘T’ in your DNA (+) strand,
then there will be a ‘U’ in the RNA strand.
As RNA polymerase moves downstream reading the
template strand of the DNA, ribonucleotides doing
the Four B’s bump into the RNA polymerase. When
the correct complementary ribonucleotide (Table
4-2) bumps the correct DNA nucleotide inside the
RNA polymerase (e.g., A-U or G-C or T-A) it will bind
and trigger a chemical reaction burst, permanently
attaching itself to the growing string of RNA (Figure
4-29).
If an incorrect match occurs (e.g., A-G), then the
ribonucleotide will bump out. Eventually, a correct
ribonucleotide will bump in, bind strongly, and
trigger the reaction. When this chemical reaction
happens, it also propels the RNA polymerase to move
further down the DNA strand to the next nucleotide.
The RNA polymerase moves at about 50 ribonucleo-
tides per second - that’s fast! Can you do the following
exercise just as fast? Complete Table 4-3 in less than
one second!
Be the cell machinery! Breakout Exercise
There’s been a lot to take in! Have a look at Table 4-2 and be the RNA polymerase! Complete Table 4-3.
Table 4-2. Transcription cipher - Nucleotide pairing table
DNA + leading strand DNA- template strand RNA
A T A
T A U
C G C
G C G
Table 4-3. Be the RNA Polymerase
5’ 3’
Leading
DNA (+)
c a t g c g t g c a a a a c c c a t g a a c c g c t g g c g a a c g a a a c c
Template
DNA (-)RNA
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113Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Figure 4-29. As RNA polymerase travels downstream un-
zipping the double-stranded DNA, it “reads” the (-) template
strand. Free-oating, complimentary ribonucleotides (C-G or
U-A or T-A) will bind strongly to the nucleotide of the DNA’s
template strand within the polymerase. This triggers the poly-
merase to attach the ribonucleotide to the growing RNA strand.
If a nucleotide doesn’t bind strongly (for example a C-A), it will
bump out of the RNA polymerase. Eventually, the correct one
will enter and be added to the growing string of RNA.
Leading (+)
Template (-)
A, U, C, G
Ribonucleotides
RNA
A
G
A
T
G
C
G
C
A
A
G
C
3’
G
T
ATT CGC
T
C
U
U
A
U
C
U
GCU
G
U
C
U
Stopping transcription
How does the RNA polymerase know when to stop
transcribing a gene? In E. coli, RNA polymerase will
stop transcribing a DNA sequence in one of three ways:
•
The RNA polymerase ‘slips off’ the DNA: If you
look back to Chapter 1, where you learned about
the structure of double-stranded DNA and the
zippering that occurs between complementary
nucleotides, you’ll notice in Figure 1-17 that there
are a different number of bonds (dashed lines)
between a G-C pair compared to an A-T pair. A
‘G-C’ complement has three bonds that hold the
complementary nucleotides together. An ‘A-T’
pair, has only two bonds. More bonds mean stron-
ger interactions, which means that the bonding
strength between A-T is weaker than G-C.
When RNA polymerase is riding along transcribing
DNA, it uses the bonds between the transcribed
RNA and the DNA (-) template strand to hold itself
connected to the DNA. A long stretch of repeat T’s in
the DNA’s (+) leading strand (which correspond to A’s
in the (-) template strand) results in a string of U’s in
RNA
(U-U-U-U-U...),
each of which also only has two
bonds. This results in weak interactions between
the RNA polymerase and the DNA, and often the
RNA polymerase simply slips off. You will often nd
stretches of T’s in DNA (U’s in RNA) at the end of a
gene. These are placed to cause the RNA polymerase
to fall off of the DNA and stop transcription
.
•
The RNA strand folds up, causing RNA polymerase
to fall off - a ‘terminator’:
What would happen
if ribonucleotides of an RNA string were able to
interact with other ribonucleotides in the same
string? You’ve seen that two different strands of
DNA can come together to form a double helix. Can
something similar happen with RNA? Yes!
Because RNA transcripts are quite exible, they
can flip and flop around, allowing nucleotides
of the RNA strand to come into contact with one
another. A string of RNA can interact with itself,
and similar rules apply: A binds to U, G binds to C.
When this happens, a ‘hairpin structure’ can form.
In the example in Figure 4-30, you’ll see what is
called the bacteriophage 82 late gene terminator.
Two regions of the RNA transcript complement
well enough to form what is called a stem and loop.
The overall structure is called a hairpin, and the
formation of the hairpin structure creates physical
stress between the RNA transcript in the RNA poly-
merase. This causes the RNA polymerase to fall off
of the DNA. In most cases, there is also a poly-uri-
dine, also called poly-U, segment (U-U-U-U-U...)
immediately following the stem. This results in
the RNA polymerase slipping off of the DNA, as you
saw in the rst example.
Figure 4-30. A hairpin is a structure where RNA folds upon
itself to create a structure that causes the RNA polymerase to
get jammed up and detach from the DNA. Another name for
this is a ‘terminator’.
UAA
CCAAAUUCAA
UUUCUGUUUCUGGGCGGU
U A
Hairpin
Uridine repeat
Loop
Stem
UAG
G C
G C
A U
G U
C G
U G
C G
G C
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114 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Figure 4-31.
A protein called Rho is able to bind to RNA tran-
scripts. As RNA polymerase is transcribing, Rho will glide up
the transcript. When it reaches the RNA polymerase, it can
‘tug’ the RNA out of the polymerase, which stops transcription.
Rho
Downstream
DNA
RNA
RNAP
• Another protein chases RNA polymerase off the
strand: Rho is a protein that has the function of
actively stopping transcription from happening.
The way Rho works is really cool. As RNA poly-
merase continues to move downstream and
transcribe RNA from DNA, Rho is able to bind near
the 5’ end of the new RNA transcript and move
downstream on the strand toward the 3’ end as if it
is chasing the RNA polymerase (Figure 4-31). The
Rho protein eventually catches up to the RNA poly-
merase and is thought to tug the RNA strand out of
the RNA polymerase, resulting in the termination
of that transcript.
What can you do with RNA?
You’ve now learned what it takes to start, do, and stop
transcription! So, what can you do with RNA?
RNA has many known functions, and many more
will be discovered in the coming decades. The most
understood use of RNA is the subject of Chapter 5,
where we will look at how RNA is read by cellular
machinery and translated into proteins. Proteins
make up the vast majority of cellular machinery that
catalyze chemical reactions or form cell structures.
RNA itself can also cause chemical reactions. ‘Ribo-
zymes’ are short strands of RNA that fold in the right
way so they can cause chemical reactions to happen.
Maybe you have heard of CRISPR-Cas9? An integral
part of the CRISPR system working includes using
RNA to guide a protein to a specic DNA sequence.
There are many more functions and uses for RNA, and
you are now well-equipped to explore further! This is
a topic of great interest amongst genetic engineers
and you will undoubtedly be able to discover many
resources by exploring the web.
What is life without RNA polymerase? Web Search Breakout
As you now know, RNA polymerase is an extremely important enzyme that is essential for life. What would
happen if RNA polymerase couldn’t function in your cells?
If you look into nature, you might run into a very poisonous mushroom called the “death cap”. This mush-
room looks like an ordinary mushroom but has a slight green color. The death cap mushroom naturally
microfactures a molecule called an amatoxin. Amatoxin is able to bind to RNA polymerase and slll-
loooooooooooww down its function. Rather than being able to transcribe thousands of ribonucleotides
per minute, RNA polymerase can only assemble a few ribonucleotides per minute. This means that cells
with amatoxin in them have a hard time completing the Three Steps to Microfacturing - which is a big prob-
lem for cell operation.
Visit amino.bio/mushroom for more information about the death cap mushroom.
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115Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Be the cell machinery! Bidirectionally! Breakout Exercise
You have recently learned that RNA polymerase can transcribe from both strands of a DNA helix. In Figure 4-28 of Going Deeper 4-10,
you can see that genetic engineers will design their DNA in a way that genes will be transcribed in opposite directions from differing
strands in order to prevent one gene from reading into the next.
In this Breakout Exercise, you will nd a DNA sequence across the middle of the table. Your goal is to be an RNA polymerase and correctly
transcribe the DNA sequence using the knowledge you’ve learned. Some tips include:
• keep an eye on the 5’ phosphate and 3’ hydroxyl ends of the DNA as these will help you know which direction to transcribe
• recall what an RNA polymerase transcribes from DNA (e.g. does it transcribe the promoter region?)
• recall which strand the polymerase “reads” in order to create the RNA transcript (e.g. the (+) or (-) strand?), and that the (+) and (-)
strand designations depend on which strand you’re reading
• recall the DNA to RNA cipher
A very important take-away from this exercise is that the RNA transcripts that are generated from each DNA strand are unique! This
means that the cell can create different RNA from the same strand of DNA if it is read in opposite directions!
You’ll also see that there is space available to complete the cell process called translation (the protein rows). This is covered in Chapter
5, and you’ll be able to come back and nish this table once you’ve learned more about how the ribosome is able to “read” an RNA tran-
script and create a chain of amino acids, also called a protein.
PROMOTER* RBS* PROTEIN CODING
N-Terminal (Protein) C-terminal (protein)
5'P (RNA) 3’OH (RNA)
5’P (DNA) A T C T A A C G A T G G C T G T A C A T T T G T A A G C 3’OH (DNA)
3’OH (DNA)
GT T T TA A AC CCACA T G G GT AAACA
TTCG
5’P (DNA)
3’OH (RNA)
5’P (RNA)
C-terminal (protein)
N-terminal (Protein)
PROTEIN CODING
————>
RBS*PROMOTER*
Table 4-2. Bidirectional transcription and translation
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