NUCLEIC ACIDS

3. Suppose that the phosphodiester backbone of DNA was replaced by amide linkages
between the sugars. How might this change affect the stability of the duplex? (Consider
charge between repulsions and chain flexibility.)
❖ To improve enzymatic stability, DNA phosphodiesters are replaced with non-ionic links.
Amides became popular because they were simple to synthesize via peptide-type
couplings. Depending on the sequence, the amides in DNA were discovered to
destabilize DNA–RNA heteroduplexes per modification. One of the elements that
controls the thermodynamic stability of nucleic acid secondary structures is electrostatic
repulsion between negatively charged phosphates; neutral or positively charged
counterparts should bind more strongly with complementary DNA or RNA. Replacement
of the phosphodiester backbone results in charge-neutral sulfonamide antisense
oligonucleotides, which resulted in a minor destabilization of the DNA-RNA duplex
compared to a DNA-DNA duplex.
https://orb.binghamton.edu/cgi/viewcontent.cgi?article=1009&context=chem_fac

4. Which process would take place more rapidly: folding a self-complementary single-
stranded molecule into a hairpin structure or joining two separate, complementary
strands into a duplex? Assume that the same number of base pairs are formed in the two
cases that the concentration of strands is equal in both cases.
● The process that takes place more rapidly is the hairpin structure whereas, this plays an
important role in replication, recognition of the origins of transfer in conjugate elements,
and the transcription of regulation. Since hairpins evolved to machinery like in other
instances, the protein has been evolved.
● Because of the large number of replicating elements, replication of E. coli has been
included. The use of DNA hairpins is ubiquitous in prokaryotes and viruses and implies
DNA cruciform has been demonstrated. Also mentioning that, single-stranded phages
have been also found to use the DNA hairpins in almost every step of their life cycle.
● With this case, Hairpin recognition can be seen as a way for living systems to expand
the repertoire of information storage in DNA beyond the primary base sequence. These
hairpin recognition examples illustrate how DNA can carry information via its
conformation.

7. Which would be more sensitive to changes in salt concentration: melting of a duplex

DNA or melting of a triplex? Why?

Like the DNA duplex, the formation of the DNA triplex is a reversible process that is
affected by both temperature and salt concentration. However, compared to the duplex, the
stability of the triplex is much more sensitive to salt concentration. Although triple helices form
with considerable sequence specificity, they are generally much less stable than their duplex
counterparts. This is mainly because they require the assembly of three polyanionic strands and
their formation is critically dependent on the ionic strength. High concentrations of monovalent
ions or lower concentrations of divalent ions, especially magnesium, promote parallel triplex
formation. Although monovalent cations stabilize triplexes, they can compete with magnesium
and thereby reduce its stabilizing effect. This inhibition can be reversed by raising the
magnesium concentration. For solutions that contain more than one type of cation the effect of
each ion will depend on its valency. It has been suggested that the cation requirement of triplex
formation depends on the T·AT and C+·GC content and that the effect of ionic strength
decreases with increasing C+·GC content. Triplexes with third strands rich in thiamine tend to
be more sensitive to salt concentrations than those rich in cytosines. In supercoiled plasmids,
which form intramolecular triplexes (H‐DNA), magnesium promotes a switch between the
conformation containing C+·GC triplets to the one containing G·GC triplets.

NUCLEOTIDE METABOLISM

1. Why would the loss of the activity of PRPP synthetase interfere with both histidine
biosynthesis and purine biosynthesis?
(references:https://medlineplus.gov/genetics/gene/prps1/ , https://www.news-
medical.net/life-sciences/Histidine-Metabolism.aspx
)

Answer: Phosphoribosyl pyrophosphate is synthesized by PRPP synthetase, with the loss of

activity of PRPP synthetase, there will be a deficiency of PRPP, A PRPP is involved in making
purine and pyrimidine nucleotides. These nucleotides are building blocks of DNA, its chemical
cousin RNA, and molecules such as ATP that serve as energy in the cell.

Histidine biosynthesis begins with ATP and Phosphoribosyl pyrophosphate , Five of histidine’s 6
Carbon atoms originate from 5-phosphoribosyl-alpha-pyrophosphate (PRPP), with the lost of
PRPP, The first step of histidine biosynthesis, which is condensation of ATP and PRPP to PR-
ATP will not occur as there is lacking of PRPP.

5. The malarial parasite Plasmodium falciparum lacks the ability to synthesize purines de
novo and depends on the purine salvage pathway for growth inside the host’s cells. The
parasite cannot salvage pyrimidine nucleotides, however, but rather must make them all
de novo. The enzyme dihydro-orotate dehydrogenase catalyzes the reduction of orotate
tp dihydro-orotate Why would medicinal chemists focus on this enzyme as a target for
the development of malarial drugs?

Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that

catalyzes the fourth reaction of pyrimidine de-novo synthesis. Pyrimidine bases are essential for
cellular metabolism and cell growth, and are considered as important precursors used in DNA
(thymine and cytosine), RNA (uracil and cytosine), glycoproteins and phospholipids
biosynthesis. The significance of pyrimidines biosynthesis in DNA and RNA makes them ideal
targets for pharmacological intervention. Inhibitors of DHODH have proven efficacy for the
treatment of malaria, autoimmune diseases, cancer, rheumatoid arthritis and psoriasis.

Reference: https://pubmed.ncbi.nlm.nih.gov/21861807/
9. The committed step in purine biosynthesis is the conversion of PRPP into
phosphoribosylamine. The nucleotides IMP, AMP, and GMP are all inhibitors of the
enzyme (glutamine-PRPP amidotransferase) catalyzing this step. Explain this pattern of
inhibition in terms of cellular efficiency.

In the purine salvage, HPRT catalyzes the conversion of hypoxanthine and guanine to IMP and
GMP respectively and adenine phosphoribosyltransferase (APRT) catalyzes the conversion of
adenine to AMP. The de novo purine synthesis pathway requires several moles of ATP for the
generation of each mole of purine nucleotide product, while HPRT and APRT require one ATP.
Much of the cellular energy is conserved in the purine salvage in comparison with the de novo
purine synthesis pathway and 90% of free purines generated during intracellular metabolism are
recycled rather than degraded or excreted.

DNA, RNA, and PROTEINS

6. Certain strains of the common gut bacterium E. coli can produce a toxin called
colicin E3, which inhibits the growth of other bacteria by enzymatically cleaving their
165 ribosomal RNA. Trace the consequences for the target bacterium upon its
encountering and taking up a molecule of colicin E3.

Answer: It will stifle or inhibit the process of translation. The cytotoxin colicin E3 binds to the
30S subunit of bacterial ribosomes and cleaves 16S rRNA near the decoding center, thus
stopping translation. The cleavage primarily impacts the elongation step. Because of the
altered decoding events, colicin E3 cleavage has an inhibitory effect. It leads to poor
occupancy of the ribosome's A site and, as a result, lower elongating peptide production.

7. The DNA and RNA polymerases of viruses are typically more prone to making
errors in polymerization than are regular cellular polymerases. Why would viruses be
more tolerant of errors than are cells?

DNA and RNA polymerases of viruses are highly prone to error since a higher rate of
mutation is associated with more error. Many polymerases in viruses, in general, lack
proofreading activity and have low reproductive fidelity. This leads to more mistakes and, as a
result, more mutations. This is more severe in the case of RNA viruses than in DNA viruses.
While normal cells have a low rate of mutation and a slow evolution rate. Hence, normal cells
are not tolerant to error and viruses are more tolerant to high error because it gives them an
adaptive evolutionary advantage. It may evolve to interact with the conserved and essential
proteins of their hosts because targeting those proteins makes it easier for the viruses to infect
all individuals within a species. This may also help the viruses to infect individuals of a new
species.