139Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Stopping Translation
How does the ribosome know when to stop translating
the RNA transcript? It is quite simple; at the end of
the coding region, there is a stop codon (Figure 5-18).
If you look back at Table 5-2, you’ll notice that three
of the sixty-four codons are marked as “Stop”. These
are codons that do not bind to tRNA molecules but
instead bind to release factors. Release factors are
proteins that are able to recognize the specic codons
in the RNA transcript causing the amino acid chain to
become disconnected and “fall out” of the ribosome.
When this occurs translation stops!
Choosing codons when designing DNA Pro-tip
If you’re designing DNA for your own biotechnology project, you might be asking the question: Which
codons do I use? There are often several codons for the same amino acid, so how do I know which one is
best. Do I get to choose whichever I want?
The short answer is that you base your decision on which host organism you plan to express your protein
in. For example, if you choose K12 E. coli bacteria, like the ones you have been using in your hands-on
experiments, you will choose codons based on which codons K12 E. coli have evolved to use.
As you learned in Chapter 4 and this chapter, the end goal of expressing a gene is not always to get proteins.
Rather, there are genes that express extremely important RNA molecules that have no intention of being
read by a ribosome - tRNA is a perfect example.
tRNAs are not expressed equally in cells, rather, there is a diversity of tRNA expression within a single
organism, and between species of organisms. For example, if we were to extract and purify all the tRNAs
listed in Table 5-2, we would not see them in equal proportions. Rather, E. coli cells express some tRNAs
more than others. See E. coli Codon Use Table below (Table 5-3).
In Table 5-3 you will see the codons separated by the amino acid they code for in alphabetical order. If you
look at “A”, which stands for alanine, you see four different codons, all of which will cause the ribosome to
add an alanine to the amino acid chain. You’ll notice that of the four codons, GCG is used the most (33% of
the time), whereas GCC, GCA, and GCU are used less so at 26%, 23%, and 18%, respectively. As a general
rule, the codon use generally reects the amount of tRNAs that are expressed in the cell. In the case of
alanine tRNAs, the CGC (anti-codon) tRNA will most highly expressed, while the CGA (anti-codon) tRNA
will be least expressed.
Based on this knowledge you can design your DNA so that the overall codon use in your gene are consistent
with the codon table. If you don’t follow this general design rule, and say you only use the GCU codon (18%),
there may be too few alanine tRNAs available during translation of your proteins. Recall back in Chapter
4: The number of molecules available for a chemical reaction is an important part of the Four B’s and can
determine whether a reaction will or will not occur. The low number of available CGA tRNAs means the
Four B’s cannot do the job and the ribosome can simply stop translation.
Designing DNA to have the right codons seems like a lot of work, right!? Nowadays you can nd “codon
optimization” software on the web, and/or when you order DNA from a DNA synthesis company, they will
automatically optimize the DNA sequence based on your target host.
As a nal note, you might now be asking, why is Table 5-3 a codon table and not an anti-codon table? The
answer to this will lead you down a rabbit hole of information that goes beyond the scope of this book. If
you’d like to follow this thread, search for “all anti-codons in E. coli”, and/or “wobble base pairs”.
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140 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Table 5-5. Be the RNA Polymerase & the Ribosome
5’ 3’
Leading DNA (+) gat gaa tgc att ccg cat gaa cgc gat aac gcg ccc gcg ggc gag
Template DNA (-)
RNA
Protein
3 6
Table 5-3. Codon usage in E. coli (alphabetical by single letter amino acid abbreviation)
Codon
Amino
acid
Codon
fraction for
same amino
acid
Fraction
of total
codon use
(%)*
Codon
Amino
acid
Codon
fraction for
same amino
acid
Fraction
of total
codon use
(%)*
UAA STOP 61% 0.2% AUG M 100.00% 2.64%
UAG STOP 9% 0.03% AAU N 49% 2.06%
UGA STOP 30% 0.1% AAC N 51% 2.14%
GCU A 18% 1.71% CCU P 18% 0.75%
GCC A 26% 2.42% CCC P 13% 0.54%
GCA A 23% 2.12% CCA P 20% 0.86%
GCG A 33% 3.01% CCG P 49% 2.09%
UGU C 46% 0.52% CAA Q 34% 1.46%
UGC C 54% 0.61% CAG Q 66% 2.84%
GAU D 63% 3.27% CGU R 36% 2%
GAC D 37% 1.92% CGC R 36% 1.97%
GAA E 68% 3.91% CGA R 7% 0.38%
GAG E 32% 1.87% CGG R 11% 0.59%
UUU F 58% 2.21% AGA R 7% 0.36%
UUC F 42% 1.6% AGG R 4% 0.21%
GGU G 35% 2.55% AGU S 16% 0.99%
GGC G 37% 2.71% AGC S 25% 1.52%
GGA G 13% 0.95% UCU S 17% 1.04%
GGG G 15% 1.13% UCC S 15% 0.91%
CAU H 57% 1.25% UCA S 14% 0.89%
CAC H 43% 0.93% UCG S 14% 0.85%
AUU I 49% 2.98% ACU T 19% 1.03%
AUC I 39% 2.37% ACC T 40% 2.2%
AUA I 11% 0.68% ACA T 17% 0.93%
AAA K 74% 3.53% ACG T 25% 1.37%
AAG K 26% 1.24% GUU V 28% 1.98%
CUU L 12% 1.19% GUC V 20.00% 1.43%
CUC L 10.00% 1.02% GUA V 17% 1.16%
CUA L 4% 0.42% GUG V 35% 2.44%
CUG L 47% 4.84% UGG W 100% 1.39%
UUA L 14% 1.43% UAU Y 59% 1.75%
UUG L 13% 1.3% UAC Y 41% 1.22%
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141Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Translation, transcription, RNA Going Deeper 5-7
Translation is more precise than transcription: During translation, there is a very specic start point
(start codon) and a very specic stop point (stop codon) in every RNA sequence. Remember that transcrip-
tion, on the other hand, is much more ‘sloppy’. The promoter region in DNA facilitates binding of RNA
polymerase. As long as the RNA polymerase can create the initiation sequence and escape the promoter, it
begins transcribing ‘roughly’ where it binds at the promoter. Transcription completes in a sloppy manner
by rho-dependent factors chasing RNA polymerase and knocking it off, or by rho-independent factors such
as a ‘hairpin’ and/or a poly-U repeat region causing the RNA polymerase to fall off the DNA.
Why not program directly in RNA? Going Deeper 5-8
Why do genetic engineers program in DNA when ultimately the cells read RNA to make their desired proteins?
While RNA is the programming language cells actively use to make proteins, it is neither very stable nor dura-
ble. DNA, however, is excellent for long-term information storage as it is very stable. Remember that one of
the main differences between DNA and RNA is the sugar present in the molecules; while RNA is made up of
ribose sugar, DNA is made up of deoxyribose. Deoxyribose is simply ribose with one less OH group. This lack
of OH aids the stability and durability of the DNA because that extra OH group makes the RNA molecule more
susceptible to hydrolysis. Hydrolysis is a process during which a molecule breaks down from a reaction with
water. While this does make RNA a less stable “programming language” for Genetic Engineers, some have
started investigating and using RNA hydrolysis as a feature in their design.
Be the cell machinery! Decode the message Breakout Exercise
Your turn! Decode the secret message from DNA with the two ciphers you learned in Chapter 4 and 5.
Table 5-4. 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 5-5. Be the RNA Polymerase & the Ribosome
5’ 3’
Leading DNA (+) gat gaa tgc att ccg cat gaa cgc gat aac gcg ccc gcg ggc gag
Template DNA (-)
RNA
Protein
3 6
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142 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Summary and What’s Next?
Congratulations! In this chapter, you not only geneti-
cally engineered the microorganism E. coli, cultured it
in a selective LB agar plate, lysed the cells, extracted
and sterilized the protein that you engineered E. coli to
microfacture, you also took your understanding of the
Three steps of microfacturing further! This is a massive
accomplishment! These are foundational skills and
knowledge that every genetic engineer needs to know
to engineer and manipulate cells.
You can see that from a DNA molecule with a promoter,
RNA is transcribed with the help of sigma factors and
RNA polymerase. If that RNA transcript molecule has
an RBS, initiation factors in the cell can interact with
the transcript and a ribosome. The ribosome uses its
rRNA to lock into the RBS and commences transla-
tion with the help of tRNAs and the initiation factors
holding an fMet. tRNAs complement the codon triplets
in the RNA transcript to a specic amino acid. As the
ribosome moves downstream on the RNA transcript,
amino acids are bound to a growing chain of amino
acids. Once the ribosome hits the stop codon, the
peptide chain is released, and it can nish folding into
a three-dimensional shape using chemical bonding, a
topic of Chapter 6. This is how and why there are now
many beautiful three-dimensional proteins oating in
the cells you engineered.
In Chapter 6, you’ll go through Step 3 of the Three
Steps of Microfacturing: Enzyme Processing. Enzyme
processing is not always necessary. In many cases,
the product of translation is a protein that is itself the
desired product. For example, you’ve created a color
protein pigment. The function of that protein is to be
colorful, and that’s it! Microfacturing stops here. In
many other instances, however, the protein created
from translation is an enzyme that is meant to be used
to cause chemical reactions to happen. In Chapter 6,
you’re going to learn how you can engineer cells to
create an enzyme that can catalyze chemical reactions
you can then use to your benet.
There’s more to translation... Web Search Breakout
The mechanism that the ribosome completes during translation is slightly more complex than described
in this chapter. If you’re keen to learn the full story, search the web for videos about this subject. Search
“RNA translation” or “EPA sites ribosome”.
Be the cell machinery! Bidirectional translation Breakout Exercise
Now that you have learned more about how the ribosome translates an RNA transcript into protein using the
RNA to protein cipher, head back to page 120 (Ch. 4) to nish the bidirectional translation Breakout Exercise.
Similar to how you found that the RNA transcripts had different sequences when transcribed from the DNA
strand in opposite directions, you’ll also nd that the protein sequences are different. Other details to note:
• the presence of starter methionines
• stop codons
• recall where translation starts (e.g. do you translate the RBS?)
While in this exercise your proteins are only ve amino acids long (called a peptide), in many real genetic
engineering scenarios, your DNA sequence would be hundreds or thousands of deoxyribonucleotides
long, leading to an RNA transcript that is hundreds or thousands of ribonucleotides long, and ultimately
an amino acid sequence (protein), that is hundreds to thousands of amino acids long. For example, the
colourful proteins that you engineered your K12 E. coli cells to produce in Chapter 4, and then extracted in
this chapter:
•
have a DNA sequence, including promoter, that is ~1000 deoxyribonucleotides (or basepairs/ bps) long
• have an RNA sequence, including the RBS, that is ~800 ribonucleotides long
• have an amino acid sequence that is ~250 amino acids long
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143Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Review Questions
Hands-on Exercise
1. Why is it important to have fresh colonies for culturing?
2. Why should you label plates?
3. How is the double streak method different from normal streaking?
4. What is the active ingredient in the Lysis Accelerator? Where does it come from? How does it help
during cell lysis?
5. What is a pellet?
6. Why should you lter sterilize your extracted sample?
7. What is “balancing a centrifuge” and why is it important?
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