Running head: DNA & RNA 1

DNA & RNA

Delia Garcia

GRT-1 Biochemistry

February 8. 2015

Julie Thompson
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DNA & RNA TASK 1

DNA is the foundation of life; it provides a blueprint at the molecular level of exactly

who and what living things are. DNA is what makes a human cell become a human, a plant cell

becomes a plant, and an animal cell becomes an animal and so on. DNA is the recipe for the

ingredients which produce a living being when combined. DNA is the highly complex, genetic

molecule located within the nucleus of a cell and contains a total of 46 chromosomes in each

molecule. The structure of which resembles a spiral stair case, or a twisted ladder. In comparison

to the previous examples, a spiral staircase has hand railing on either side with the steps located

in the center. DNA it is comprised of a backbone on either side or nucleotide base pairs in the

center.

DNA Structure

The DNA Backbone is a made of a bond which occurs between a five carbon sugar

(Pentose), and a phosphate group. The DNA Backbone however, runs in different directions

chemically on either side. One side begins connected to the sugar molecules fifth carbon and

ending in the position the next phosphate would take at the sugars third carbon. This pattern is

denoted 5 to 3. On the other side, the pattern is in reverse connecting to the sugars third carbon

and ending at the sugars fifth carbon as 3 to 5.The nucleotide base pairs consists of one of four

nitrogenous bases which bond to the correct corresponding nucleotide to form a polynucleotide.

The four nitrogenous bases include Adenine (A), Thymine (T), Guanine (G), and Cytosine (C).

Like a magnet bonds a positive charge only to a negative charge, (A) can only bond to (T) while

(G) and (C) can only bond to one another.

DNA Replication Process

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DNA replication occurs for many reasons, it is the factor behind hair and nail growth and

why wounds are able to heal. Anytime there is growth in the body, the DNA replicates; this is

because every cell in the body has to go through the cell cycle to produce more. The cell cycle

requires that the entire DNA within the nucleus copy itself so the next cell produced is exactly

the same. The entire replication process entails replication, transcription, and translation.

In order to describe the function of the enzymes in DNA replication, visualization of a zipper

provides an easy to understand point of comparison. A zipper has teeth on either side which

interlock and a sliding mechanism which moves to separate.

DNA Replication Enzymatic Functions/Actions

DNA Helicase

DNA Helicase is the molecular motor-protein which separates the hydrogen bonds

between the nucleotide base pairs so that a single-stranded DNA for replication, transcription,

and translation. Single strand binding proteins adhere to the strands to keep the strands from

becoming reattached. In the previous example, DNA Helicase represents the slider mechanism

of the zipper while the interlocking teeth depict the corresponding base pairs. What the sider

does is open and close the teeth on a zipper, therefore, the helicase opens and closes the

nucleotide bond of the template strands of DNA using energy gleaned from ATP hydrolysis.

The point of the separation caused by Helicase is called the replication fork, and the

unwound strands can now which be used as template to replicate the DNA are known as the

leading strand and the lagging strand. The leading strand is the on the top runs 5 to 3 while and

the lagging strand is on the bottom and runs in 3 to 5.

DNA polymerase I & III

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DNA polymerase I and III each act on upon the template strands differently, yet both

enzymes will exhibit some similar characteristics in their functional ability. DNA polymerase III

is a critical element in DNA reproduction because of its ability to supplement the missing

nucleotides upon the template strand; while DNA polymerase I adds the properly matching

nucleotides, it also serves to eliminate the RNA primers from a previously fragmented segment.

It is important to mention that the two are not interchangeable; even though DNA

polymerase I, II, and III all have 3 to 5 directional exonuclease activity and synthesize DNA in

the 5 to 3 direction; they are all, by definition, moderately dissimilar. Visualization of the

unique traits can made clear through the concept of workplace diversity; similar to factory

employees, each has their own skill set unique to their individual job description.

Enzymes that possess nuclease activity have the ability to degrade nucleic acids.

Exonuclease activity refers to the ability to make cuts on the end of the DNA strand, while

endonuclease refers to the interior cleavage of a strand. Therefore, all DNA polymerase are

capable of erasing a mistake in the backwards direction (3 to 5), acting upon the end of a

DNA strand. As such both Polymerases I and III share this proof reading capacity wherein

errors can be cut off the end of the strand through exonuclease activity.

Essentially, all DNA polymerases assist in transporting chromosomal data from one

generation to the next by means of the DNA replication process, yet the subtle differences

produce widely different outcomes in the process. For instance, DNA Polymerase III has the

highest processivity meaning that it has the ability to incorporate the highest number of dNTPs

before falling away from the strand.

It is true that all polymerases have the ability of adding nucleotides, yet none produce the

addition volume or staying capacity of DNA polymerase III. Polymerase III is generally able to
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add roughly 500,000 dNTPs before falling away; while DNA Polymerase I, on the other hand,

can only add approximately 200 dNTPs before it falls away from the complex(Moof University,

2013). Like the Marvel Superheros, each of the polymerases has a unique superpower, for

Polymerase III it is processivity for DNA polymerase I its 5 to 3 exonuclease activity.

RNA Primase, RNA Primer, DNA Primase

The determination of which strand is either the leading or lagging strand can assist in the

appropriate identification of Polymerase I and III. The strand which continues on in the 5 to 3

direction is known as the leading strand while the strand in now in the 3 to 5 direction is called

the lagging strand. Interesting aspect surrounding these strand designations is the template strand

directionality is not the definite factor in why one is leading and the other lagging, this has to do

with the mechanisms of action in which DNA polymerase can function on the strand, basically

the difficultly in directionality is the result of polymerases inability to work the same in either

direction.

DNA Polymerase I catalyzes the template-directed polymerization of nucleotides into

duplex DNA in a 53 direction. DNA Polymerase I possesses a 35 exonuclease activity or

"proofreading" function, which lowers the error rate during DNA replication, and also contains a

53 exonuclease activity, which enables the enzyme to replace nucleotides in the growing

strand of DNA by nick translation(Promega Corporation, 2015). Primase catalyzes the synthesis

of an undersized RNA segment called a primer. Primase has significance in the replication of

DNA as DNA polymerases are unable to instigate synthesis without and the presence of RNA or

DNA primer during the elongation phase. This inability occurs because DNA polymerases are

inept in the melting of duplex DNA (disrupt hydrogen bonds) in order to separate the two strands

to be copied; all DNA polymerases so far discovered can only elongate a preexisting DNA or
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RNA strand, the primer; they cannot initiate chains(Lodish H, Berk A, Zipursky SL, et al,

2000).

Since Primase produces RNA molecules, the enzyme is a type of RNA polymerase.

Primase functions by synthesizing short RNA sequences that are complementary to a single-

stranded piece of DNA, which serves as its template. Since the leading strand does not require as

much energy to replicate as the lagging strand, DNA polymerase III runs along the strand adding

the appropriate nucleotides after RNA Primase lays down a starting point at the replication fork.

DNA Primase catalyzes the formation of RNA primers for DNA synthesis; however, DNA

polymerase III only requires minimal assistance from a primer to address the nucleotides near the

replication fork at the beginning of the template strand.

RNA Primase gives DNA polymerase III a point of origination in the replication process;

and it is perhaps similar to verbal directions to a new restaurant from a friend, that tells DNA

Polymerase III to go from here. Another comparative example is the painting of an interior

wall; primer is first applied in order to provide the next layer of paint a fresh area to grab onto.

The lagging strand however, requires a slightly more complex process in replication, since this

strand runs 3 to 5; replication is significantly more perplexing as it requires more energy. Thus,

DNA polymerase I begins its work removing RNA and adding nucleotides to achieve completion

of producing a new copy of DNA.

Okazaki Fragments

Since DNA polymerase I is the only polymerase with exonuclease ability in the 5 to 3

direction, and the lagging strand is 3 to 5, it works on the leading strand. On this strand, RNA

polymerase adds RNA primer which provides assistance in small fragments running along the
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template strand adding the appropriate nucleotides. Next DNA Polymerase III follows the path

RNA Primase has laid out and begins to encode the template strand.

This process repeats several times before DNA polymerase I comes in and removes the

RNA in fragmented sections and then adds the correct nucleotides. DNA ligase then will begin

its duty of connecting the Okazaki fragments to a complete strand of DNA. These fragments

were first discovered by their namesake, a married scientist couple, who found these fragments

which allow DNA polymerase I to ultimately duplicate DNA appropriately where DNA

Polymerase III obligates a mitigating its deficit in the accommodation for the change in

directionality.

DNA Ligase

Ligase is the enzyme which ultimately produces a covalent bond between the neighboring

DNA fragments. It can be used to produce ligation or bonds by either blunt or cohesive ends to

form the recumbent DNA strand. DNA Ligase catalyzes a phosphodiester bond between the

juxtaposed 5 phosphate group and 3 hydroxyl terminal in double stranded DNA (New England

Bio labs, 2014). The process of DNA Ligation occurs in three phases. First, a reaction occurs to

produce self-adenylation with either free ATP or NAD. Next, the newly formed adenylated group

will move to 5 prime end of the parent strand and form a bond with the phosphorylated end. The

final phase occurs when a reaction between the 5 adenylated end and the 3 hydroxyl end to

produce the covalent bond through the release of AMD.

RNA polymerase

RNA polymerase binds the template DNA and contributes high processivity to enzymes.

RNA Polymerase has an identical nucleophilic attack of DNA Polymerase does on the 3 OH of

the incoming NTP, as opposed to the 5 end; and then synthesizes in the 5to 3 direction. DNA
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enters active site and the strands separate. Once inside the RNA polymerase, the template strand

will then take a right angle turn and the clap will close tightly over the template strand. It does

not let go of the template strand as it continues transcribing and moving from one nucleotide to

the next.

The bridge oscillates between straight and curved to ratchet the template to move to the

next nucleotide and ensures nucleotides are not skipped in the process. The Rudder (protein loop)

assists in separation of DNA and RNA. The RNA has an ability to proofread and stop synthesis

and backtrack when errors occur. Transcription factor, TFIIS has the ability to correct the errors

RNA polymerase signals to and allows RNA polymerase to begin the process again.

Nucleotide triphosphate substrates enter the RNA polymerase through a pore and then the

nucleotides hydrogen bond with the templates. This is how RNA polymerase functions correctly

by adding the proper nucleotide onto the growing RNA chain. The correct rNTP causes a loop of

protein to enclose and ensures accuracy in transcription. Catalysis is the temporary hybrid helix

formed of DNA and RNA which occurs so that during transcription synthesis of mRNA is not

problematic due to a permanent bond between the two.

RNA polymerase & the Death Cap Mushroom

RNA polymerase is also responsible for diseases of the body, Werner's Syndrome is a

rapid aging disease that starts in adolescence and causes death around 50 years old; in cells with

two bad copies of Werners protein, RNA Pol II transcription rates are half the normal rate of

3000 nucleotides per minute (St. Dominic School, n.d.). RNA polymerase is so important that

without its function death will occur, an example of this is seen when someone ingests a Death

Cap mushroom.
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According to American Mushrooms, this particular species of mycorrhizal fungi is of an

ecological benefit, living on the roots of live trees, providing phosphorus, magnesium, and other

nutrients to the tree in exchange for carbohydrates (American Mushrooms, 2006). This is slightly

similar to our own human internal processes which occur when we consume food and synthesize

proteins for energy. The Death Cap mushroom is from the Amanita phalloides family which

has quite the reputation for its highly poisonous effects on the body which also happens to occur

at the cellular level in which humans synthsize the enzyme RNA Polymerase (Autoimmune

Maven, 2010).

Out of over 100,000 mushroom species, only around 100 or so are highly toxic. The

toxicity of the Amanita species is due to the presence of two groups of toxins known as

amatoxins and phallotoxins, both multicyclic peptides; presumably, the death cap contains six

related phallotoxins and five or more amatoxins (Shiells, 2013). These peptides are not

essentially in and of themselves caustic, or have even a slight have a degree of tolerable toxicity.

Actually, the majority of mushroom poisonings are not fatal; yet when they are, it is often

attributable to the Death Cap mushroom. Consequently, the toxins present are secondary

metabolites produced in specific biochemical pathways in the fungal cells (CDC, 1997).

There are several factors which can influence the degree of poisoning caused by a

chemical, the route of entry, amount or dose, the toxicity, removal or elimination from the body,

or the biological variation(Canadian Center for Occupational Health & Safety, 2009). The death

cap mushrooms poison is not a food borne bacteria; in fact the phallotoxin elements are non-

lethal on their own, generally this peptide only causes gastrointestinal upset. The primary toxin,

alpha amaitin, however, causes fatal liver destruction in less than three days after ingestion.
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According to the St. Dominic School Alpha-amanitin reduces transcription rates to 5 or

6 nucleotides per minute and thats not enough to sustain the life of a cell (St. Dominic School,

n.d.). Since the fungi does not cause typical effects thought to occur with poisoning, symptoms

are of slow onset as the toxins assault RNA polymerase. Because RNA polymerase is an active

element in the production of mRNA, micro-RNA, and small nuclear RNA; without it, protein

synthesis cannot occur, thus terminating metabolism of the cell and ultimately resulting in

cellular death. When the body can no longer replicate new cellular tissues, death is often the

unavoidable end result.

This mushroom begins its silent attack on the liver, then the kidneys, a liver transplant is

currently the only known method of treatment and the chance of survival is often quite

diminutive. The succinct precipitate of the death cap mushroom is not a reaction to a harmful

ingested chemical, for instance, hemlock or mercury. Instead, the body dies from the inability to

function on the microcellular level. Hence, an absence of RNA polymerase renders cells

incapable of carrying out the practice of replication. Intrinsically, the ultimate cause of death

occurs in cellular demise because new cells are not regenerated when living tissues die.

DNA Transcription & Translation

Once the DNA Template strands move through the replication process they need to be

transcribed and translated into the appropriate proteins that make up cells in the body.

Transcription begins as the template strand is synthesized into RNA. RNA is similar to DNA yet

it does not have the double helix structure, it has a single sugar-phosphate backbone with a sugar

that has an extra molecule of oxygen attached; and instead of the (T) nucleotide found in DNA,

Uracil (U) takes the place of bonding with (A).

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Transcription is triggered by a promoter sequence within the strand; the sequence is at

times referred to as the TATA box because the initiation sequence begins with thymine, adenine,

thymine, and adenine. Transcription begins upstream at the 5 end with RNA polymerase copying

the DNA Sequence downstream to the 3 end of the strand until it reaches the termination signal.

Transcribing a new strand of mRNA as a result this will be used later in the translation phase.

This process is similar to copying a hand written document in a copy machine, the original is left

in-tact, while the new copy is produced-though slightly different from the original because the

copy ink (uracil) is not the same as the original ink from a pen (thymine).

Before the mRNA can exit the nucleus and begin the next phase, a cap is added to each

end the 5 cap is added to the 5 end while the Poly A tail is added at the 3 end. These caps

protect the mRNA from degradation by passing enzymes. Just before the process is complete and

mRNA is able to exit the nucleus, RNA Splicing occurs to remove any excess genetic

information not needed in the strand.

Translation, also known as protein synthesis, is the process which occurs as mRNA enters

the ribosome which then encodes the mRNA. Inside the ribosome, mRNA passes through

binding sites where tRNA translates nucleotides into amino acids and proteins. TRNA consists of

an amino acid on one end and the anti-codon at the other end, the binding site essentially scans

the mRNA in sections (triplet codon) and the tRNA adds the correct sequence of nucleotides to

the strand. The tRNA amino acid end depends on the bases found at the anti-codon, as the strand

is scanned through the ribosome, a chain of amino acids (polypeptide chain) begins to emerge

from the tRNA and in the end a protein is synthesized.

Diagrams

I. DNA Structure:
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II. RNA Structure:

III. DNA Replication, Transcription & Translation

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IV. DNA Replication, (Elongation Phase)

V. DNA Transcription
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VI. DNA Translation

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Reference

American Mushroom.com. (2004). American Mushrooms: The Death Cap. Retrieved from

http://americanmushrooms.com/deathcap.htm

Auto-immune Maven. (2010). RNA polymerase Inhibition and the Death Cap Mushroom A.K.A

Dont Eat That!! Retrieved from

https://autoimmunemaven.wordpress.com/2010/11/27/rnapolymerase-inhibition-and-the-

death-cap-mushroom-a-k-a-dont-eat-that/

Canadian Center for Occupational Health & Safety. (2009). What Makes Chemicals Poisonous.

Retrieved from http://www.ccohs.ca/oshanswers/chemicals/poisonou.html

Centre for Disease Control and Prevention (CDC) (June 1997). "Amanita phalloides mushroom

poisoning Northern California, January 1997". MMWR Morb. Mortal. Wkly. Rep. 46

(22): 48992. PMID 9194398.

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H.

Freeman; 2000. Section 12.2.The DNA Replication Machinery. Retrieved from

http://www.ncbi.nlm.nih.gov/books/NBK21751/

Moof Univesity. (2013). DNA Replication (Part 2 of 3) - DNA Polymerases - Prokaryotes and

Eukaryotes Comparison. Retrieved from https://www.youtube.com/watch?

v=92m_o3uDzzs

New England Bio labs. (2014). DNA Ligation. Retrieved from https://www.youtube.com/watch?

v=Q3xVGvEGIsg

Promega Corporation. (2015) DNA Polymerase I. Retrieved from Promegea website

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https://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-

competent-cells/dna-polymerase-i/

Shiells, E. (2009). Deadly mushroom chemistry. Chemistry World. Retrieved from

http://www.rsc.org/chemistryworld/2013/03/chemistry-mushroom-fungus

St. Dominic School. (n.d.). RNA Polymerase II: The Reader of the Secret Code!. Retrieved from

http://cbm.msoe.edu/images/contentImages/smartTeams/alumni/2007-

08/St.Dominic_RNAPol.pdf