Renee Louise S.

Casulla ABT 107- C1

Central Dogma of Molecular Biology

The central dogma of molecular biology tells us the flow on how the genetic information
in the cells from the DNA to messenger RNA (mRNA) to protein is synthesized. It is divided into
three major processes namely: replication or the process involving the synthesis of DNA,
transcription or the synthesis of RNA, and lastly translation which involves the synthesis of
protein.

Replication

The genetic material, which is the DNA, is replicated in a semi-conservative manner

wherein a newly synthesized molecule is the combination of the parent DNA’s old strand and the
newly synthesized DNA strand. The two strands of the parents DNA act as templates or
patterns from which complementary strands are synthesized. Two important features of the DNA
replication are the specific base pairing between the template and the complementary strands,
and the ability of the enzymes to proofread, which ensures the accuracy of the replication. This
process is divided further into three subprocesses:

Initiation

In the initiation phase, its main goal is to dissociate the complementary strands of
DNA into single strands which would serve as the DNA templates. By breaking the
hydrogen bonds between the complementary strands, the helicase makes it possible for
the ATP to unwind double-stranded DNA.

● Reannealing of the strands is prevented by the action of several single

strand binding proteins or SSBPs that bind to the single strand DNA.
● The created twisting tension on the DNA during unwinding is relaxed by
the enzyme DNA gyrase.
● Primase synthesizes the short segments of RNA to act as primers for
elongation of the complementary strand.
● A short segment of the DNA is complemented by the primase with RNA
nucleotide in the 5’ to 3’ direction, once the templates are open.
● The short RNA segments would act as RNA primers to give the 3’-OH
needed by DNA polymerase III to catalyze phosphodiester bond formation
during elongation when the nucleotides are added.

Elongation of the Complementary Strand

In this phase, the complementary strand which is synthesized by the 3 'to 5'
template, is stretched in the same direction as the hairpin unwinding, hence the name
leading strand. The leading strand extension requires only one primer and runs
continuously in the 5 'to 3' direction.
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On the other hand, the complementary strand of the 5 'to 3' template is also
synthesized in the 5 'to 3' direction, but against the direction of the replication fork, which
is why it is called the lagging strand.

In addition, the replication of the 5 'to 3' template requires multiple primers that
produce multiple discontinuous segments of complementary DNA called Okazaki
fragments. After that, the DNA polymerase I proofreads the DNA molecule, and gets rid
of the primers via exonuclease activity beginning from the 3` to 5` direction. It then
replaces the excised primers with DNA nucleotides however leaves a break alongside
the sugar phosphate spine of the replicated DNA fragments. These breaks are sealed
and held on together by enzymes referred to as ligase.

Termination

The process of replication finally comes to a halt once all of the parent DNA
nucleotides have been complemented. The two daughter molecules are exact replicas of
each other and of the parent DNA.

Transcription

As stated earlier, transcription involves the synthesis of RNA from a DNA template. It can
be divided into three steps:

Initiation

In the initiation phase of transcription, the RNA polymerase binds to a specific

sequence in DNA which is upstream of the gene to be transcribed, called a promoter.
This process is made possible with the help of sigma factor (o) polypeptide which
recognizes the promoter sequence, allowing RNA polymerase to selectively bind to it.
Once complete, the o factor will finally dissociate and is free again for another round of
initiation.

Elongation

RNA polymerase moves along the anti-coding (antisense) DNA strand, the 3’ to
5' strand of the gene. RNA polymerase separates the two strands while complementing
template DNA nucleotides with RNA nucleotides as it travels through the gene. Once a
segment is transcribed, the DNA strands are reconnected because the DNA to DNA
interaction is stronger than the DNA to RNA interaction. In this way, a single-stranded
RNA is produced. This proceeds until the enzyme reaches the termination point.
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Termination

When RNA polymerase reaches the termination point, the rho (p) protein factor
attaches to the enzyme and cleaves it from DNA to stop transcription. This creates RNA
that is complementary to the anti-coding strand, which can be one of three types, namely
messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). All three
types are essential for translation.

Translation

Activation of Amino Acids

Amino acids are attached to their respective tRNAs via the action of the enzyme
amino-acyl synthetase at the cost of ATP.

Initiation

The mRNA–ribosome complex is created during initiation, and the first codon
(always AUG) binds the first aminoacyltRNA (called initiator tRNA).

Initiation factor 1 (IF1) promotes the dissociation of the tiny subunits of ribosomes
before translation begins. Following the dissociation of the subunits, initiation factor 3
(IF3) attaches to the 30S subunit, preventing the 3OS and 5OS subunits from realigning,
and binding the 30S subunit to the mRNA start codon. The first aminoacyl RNA (which
carries formylated methionine or fmet) is coupled to the 30S subunit by initiation factor 2
(IF2), which has bound GTP. The initiation complex is then completed when IF1 attaches
to the 30S subunit. When the 50S subunit connects to this complex, IF3 and IF1
separate from the 30S subunit. GTP is then hydrolyzed into GDP and Pi, causing IF2 to
dissociate. This causes the entire 70S ribosome to develop, which then forms a complex
with the mRNA to be translated.

The formyl group in fmet is thought to inhibit polypeptide elongation at the amino
terminus (N-terminus). The fmet-tRNA completes the mRNA's start codon.

Elongation

The additional codons are read in order during the elongation phase, and the
polypeptide expands by adding amino acids to its C-terminal end. Once the fmet is
established at the P-site with elongation factor Tu (EFTU) and GTP, a new amino
acid-tRNA complex enters the A-site corresponding to the next codon. The 50S subunit's
peptidyl transferase catalyzes the creation of a peptide bond between the fmet and the
amino acid carried by the tRNA in the A-site. The tRNA in the P-site is then released and
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recycled in the cytoplasm. The activity of elongation factor G (EFG) and GTP causes the
ribosome to travel along the MRNA in the 5' to 3' direction, displacing the amino
acid-tRNA complex from the A-site to the P-site and freeing the A-site for the next amino
acid-tRNA complex.

The cycle of amino acid-tRNA entrance, creation of peptide bonds, and

displacement of the amino acid-tRNA complex from the a-site to the p-site is repeated
until the a-site meets a stop codon during polypeptide elongation.

Termination

When one of the stop codons, such as UAA, UAG, or UGA, is encountered by
the A site of the ribosome, one of the release factors enters the A-site and causes the
translation to halt. UAA and UAG would be recognized by release factor 1, whereas UAA
and UGA would be recognized by release factor 2 (RF2). Both RF1 and RF2 work to
break the connection between the developing polypeptide chain and the tRNA to which it
is bound, allowing the polypeptide product to be released from the ribosome. Release
factor 3 (RF3) aids RF1 or RF2 binding to the ribosome through GTP.

The ribosome dissociates and prepares the ribosomal subunits for another cycle
of translation as soon as the polypeptide is released, thanks to the activity of initiation
factor 1. Before becoming functional, the linear polypeptide chain can be folded or
further processed (for example, by adding functional groups such as sugars or
phosphates).

Overview

Figure 1. Overview of the central dogma of molecular biology.

Renee Louise S. Casulla ABT 107- C1

Importance of the Processes in Central Dogma that

was Applied in Making Recombinant Organisms

The central dogma is the concept that genetic information flows from DNA to RNA to
protein in order to function. It has three major steps and subprocesses under it which are
heavily utilized in the recombinant DNA technology. Without the flow of the central dogma, none
of the cells would be able to function at all, as it is present in every organism intrinsically,
recombinant or nonrecombinant ones.

Listed below are the different subprocesses in the central dogma that are included as the
basic steps in the creation of recombinant organisms:

● Isolation of genetic material (DNA)

Within the membrane, DNA is encased. As a result, the cell wall is broken
down in order to retrieve the genetic material. Cell wall disintegrating enzymes
such as Lysozymes for bacterial cells, cellulose for plant cells, and chitinase for
fungus cells can be used to do this.

● Cutting of DNA at specific locations

Restriction enzymes are used to cut DNA at particular recognition

sequences, resulting in the required DNA fragments.

● Joining of DNA fragment

Sticky ends ligation, blunt-end ligation, and homopolymer tailing can all be
used to create DNA fragments from a cloning vector.

● Insertion of DNA into the host cell.

Transformation, transduction, and vectorless gene transfer can all be

used to get the recombinant DNA into the host cell.

● Selection and screening of transformed cells

Any of the following approaches can be used to select and screen

transformed cells: immunological method, nucleic acid hybridization and
blue-white screening, or insertional inactivation.

As years pass by, many scientists try to create different recombinant organisms with the
utilization of the central dogma and its processes involved. But before having to achieve that,
previous scientists actually had to build a foundation first, specifically being able to properly
know the uses and applications of the different processes in the central dogma, in order to
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create new recombinant organisms. One of the recorded pioneers was the biochemist Paul
Berg, who made use of the restriction enzyme in order to cut the DNA into separated fragments
or conducted the process which was later coined as DNA splicing. By utilizing an enzyme,
particularly ligase, he was able to join two DNA strands concurrently, forming a hybrid circular
molecule. This breakthrough made him the first person to synthesize the first hybrid DNA or the
recombinant DNA (rDNA) molecule we know today. Later on, because of this gene transfer
technique that he pioneered, other scientists and researchers had been being paved the way to
create bacteria that produce substances mostly used in the field of medicine and the reason
why today, he is recognized and dubbed as the “father of genetic engineering”.

With the utilization of the gene transfer technique exemplified by Berg, another scientist
came into play and was able to produce a human protein made out of bacteria for the first time.
Herbert Boyer of UCSF introduced an insulin gene into the bacteria E. coli, synthesizing
synthetic human insulin. Not only was this experiment helpful for many people suffering from
diabetes, as the previous prices in the medication of the disease was so much expensive, but it
also opened new opportunities for other experiments involving cloning procedures and DNA
sequencing techniques. Vaccines were also invented, and even other commercially consumed
recombinant products were introduced to the market.

Golden rice rich in beta carotene, transgenic goats producing spider silk, chymosin for
our cheesemaking, low acrylamide potatoes for less patchy potatoes; these are just some of the
important and prominent commercially available recombinant organisms that are heavily used
and consumed even by normal citizens nowadays. All of these, without the utilization of the
mentioned processes involved in the central dogma, will not be possible.
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References

Aryal, S. (August 31, 2018). Prokaryotic Translation (Protein Synthesis).

https://microbenotes.com/prokaryotic-translation-protein-synthesis/

Basics of DNA Replication. (n.d).

https://courses.lumenlearning.com/boundless-biology/chapter/dna-replication/

Bhatia, S. (May 2018). History, scope and development of biotechnology.

https://iopscience.iop.org/book/978-0-7503-1299-8/chapter/bk978-0-7503-1299-8ch1

Dewei, S. (September 2014). Central Dogma of Biology.

https://www.slideserve.com/shen/central-dogma-of-biology

OpenStax, Concepts of Biology. OpenStax CNX. May 18, 2016

http://cnx.org/contents/s8Hh0oOc@9.10:2ousESf0@5/DNA-Replication

Overview of Translation. (n.d.)

https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451-biochemistry-d
efining-life-at-the-molecular-level/chapter-11-translation/

Paul Berg. (n.d.). https://www.nobelprize.org/prizes/chemistry/1980/berg/facts/

Prokaryotic Transcription. (n.d.)

https://courses.lumenlearning.com/boundless-biology/chapter/prokaryotic-transcription/

Prokaryotic Transcription. (n.d.)

https://courses.lumenlearning.com/wm-biology1/chapter/prokaryotic-transcription/#:~:text=In%2
0Summary%3A%20Prokaryotic%20Transcription,that%20assists%20only%20with%20initiation.

The Genetic Code. (n.d.).

https://courses.lumenlearning.com/boundless-biology/chapter/the-genetic-code/#:~:text=The%2
0central%20dogma%20of%20molecular,specify%20the%20sequence%20of%20proteins.