RNA World Hypothesis, Properties of DNA&RNA

3/26/2024
BBT1206: STRUCTURE AND

FUNCTION OF GENES
BOT2210: MOLECULAR STRUCTURE

AND FUNCTION OF GENES
The RNA World Hypothesis & Ribozymes;

Physical & Chemical properties of RNA & DNA
Prof. Arthur K.Tugume (B.Sc., M.Sc., PhD)
Department of Plant Sciences, Microbiology & Biotechnology,

CONAS, Makerere University
Prof Arthur Tugume, BOT2210/BBT1206
TOPICAL COURSE OUTLINE

1. Meaning of Gene, Genes, Genesis and Genetics
2. Diversity of Life forms and Macromolecules
3. DNA: Building blocks and the Double helical structure
4. RNA: Structural and Functional Diversity of the RNA world
5. Ribozymes and the RNA world Hypothesis
6. Physical and Chemical Properties of DNA and RNA
7. Cellular Packaging and Storage of DNA
8. DNA Replication: Models and Mechanisms
9. DNA Damage, Repair and Recombination
10. Transcription, Transcription Factors, and Post-transcription
11. The Genetic code and Protein synthesis
12. Gene Structure and Principles of Gene expression
13. Genes, Development and Metabolism: Control of Gene
expression in Prokaryotes
14. Genes, Development and Metabolism: Control of Gene
expression in Eukaryotes
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WHICH MOLECULE IS
“ANCESTRAL” TO OTHERS?
Prof Arthur Tugume,

BOT2210/BBT1206
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Prof Arthur Tugume,

BOT2210/BBT1206
Prof Arthur Tugume,

BOT2210/BBT1206
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Prof Arthur Tugume,

BOT2210/BBT1206
WHAT IS THE RNA WORLD

HYPOTHESIS?
 Self replicating RNA molecules were precursors to
the current life that is based on DNA, RNA and
proteins.
 It is generally accepted that the current life on
earth descends from an RNA world, although RNA-
based life may not have been the first to exist.
 RNA in modern cells is an evolutionary remnant of
the RNA world that preceded the current world.
 RNA stores genetic information like DNA and
catalyzes chemical reactions like enzymatic
proteins!!!!!
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RNA WORLD & THE ORIGIN OF

LIFE
 The discovery that RNA molecules can act as catalysts provides
a possible solution to a long-standing dilemma in the study of the
origin of life
◦ DNA encodes the genetic information of proteins but DNA replication

and transcription requires proteins. So which came first in the
evolution of life?
◦ But if RNA can serve both as a repository of information (in its

sequence of nucleotides) and as a catalyst, then it has both properties
needed for life.
◦ This provides the basis for the notion that life began as RNA — the so-
called "RNA World".
RNA world & the origin of life

 The RNA world hypothesis proposes that life based on RNA pre-
dates the current world of life based on DNA, RNA and proteins.
 RNA is able both to store genetic information, like DNA, and to

catalyze chemical reactions, like an enzyme protein. It may therefore
have supported pre-cellular life and been a major step in the
evolution of cellular life.
 Thomas Čech (2011) has argued that that multiple self-replicating

molecular systems probably preceded RNA. Proteins large enough to
self-fold and have useful activities came about only after RNA was
available to catalyze peptide ligation or amino acid polymerization,
although amino acids and short peptides were present in the earlier
mixtures.
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RNA world & the origin of life

 Hence, Čech (2011) proposes that the RNA world evolved into a
world of RNP enzymes, such as the ribosome and ribozymes,
before giving rise to the DNA, RNA and protein world of today.
 DNA is thought to have taken over the role of data storage

due to its increased stability, while proteins, through a greater
variety of monomers (amino acids), replaced RNA's role in
specialized biocatalysis. The RNA world hypothesis suggests that
RNA in modern cells is an evolutionary remnant of the RNA world
that preceded ours. Also, many critical cofactors (ATP, Acetyl-CoA,
NADH, etc) are either ribo-nucleotides or substances clearly related to
them.
IMPORTANT ENZYMATIC PROPERTIES OF

RNA FOR THE BEGINNING OF LIFE
 RNA enzymes, or ribozymes, are still found in today's DNA-based life

and could be examples of living fossils, where they play vital roles, such
as those in the ribosome, which is vital for protein synthesis.
◦ The ability to self-duplicate, or duplicate other RNA molecules.

Relatively short RNA molecules that can duplicate others have been
artificially produced in the lab.
◦ The ability to catalyze simple chemical reactions which would

enhance the creation of molecules which are building blocks of
RNA molecules—i.e., a strand of RNA which would make creating more
strands of RNA easier. Short RNA molecules with such abilities have been
artificially formed in the lab.
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IMPORTANT ENZYMATIC PROPERTIES OF

RNA FOR THE BEGINNING OF LIFE
◦ The ability to catalyze the formation of peptide bonds, in

order to produce short peptides or longer proteins. This is
done in modern cells by ribosomes, a complex of several RNA
molecules known as rRNA and many proteins. The rRNA molecules
are responsible for its enzymatic activity. A much shorter RNA
molecule has been formed in the laboratory with the ability to form
peptide bonds, and it has been suggested that rRNA has evolved
from a similar molecule. It has also been suggested that amino acids
may have initially been involved with RNA molecules as cofactors
enhancing or diversifying their enzymatic capabilities, before evolving
to more complex peptides. Similarly, tRNA is suggested to have
evolved from RNA molecules that began to catalyze amino acid
transfer.
WHY SHOULD A CELL STORE GENETIC

INFORMATION IN DNA AND NOT RNA?
 RNA is a very similar molecule to DNA, but the two have only two
chemical differences. The overall structure of RNA and DNA are
immensely similar—one strand of DNA and one of RNA can bind to form
a double helical structure. This makes the storage of genetic information in
RNA possible in a very similar way to the storage of information in DNA.
 The major difference between RNA and DNA is the sugar moiety of the
nucleotide, and the replacement of U in RNA for T in DNA.
 An OH group at the 2'-position of the ribose sugar in RNA makes RNA
less stable because when not constrained in a double helix, the 2‘-OH can
chemically attack the adjacent phosphodiester bond to cleave the
phosphodiester backbone.
 Chemically, U is similar to T, differing only by a methyl group, and its
production of U requires less energy. A will readily bind U or T. However, U
is one product of damage to C making RNA particularly susceptible to
mutations which can replace a GC base pair with a GU (wobble) or AU
base pair.
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Adenine (A) Guanine (G)
Cytosine (C) Thymine (T) Uracil (U)
WHY SHOULD A CELL STORE GENETIC

INFORMATION IN DNA AND NOT RNA?
 The chemical properties of RNA make large RNA molecules inherently

fragile, and they can easily be broken down into their constituent
nucleotides through hydrolysis. The aromatic bases also absorb strongly
in the ultraviolet region, and would have been susceptible to damage
and breakdown by background radiation.
 These limitations do not make use of RNA as an information storage

system impossible, simply energy intensive (to repair or replace
damaged RNA molecules) and prone to mutation. While this makes it
unsuitable for current 'DNA optimized' life, it may have been acceptable
for more primitive life.
 For example, a greatest portion viruses infecting plants and very many
viruses infecting animals and other life-forms still use RNA as their
genetic material, rather than DNA.
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RIBOZYMES
 A ribozyme – an RNA molecule with a well defined tertiary
structure that enables it to catalyze a chemical reaction. Ribozyme
means ribonucleic acid enzyme. It may also be called an RNA
enzyme or catalytic RNA.
 Until about 25 years ago, all known enzymes were proteins. But then it
was discovered that some RNA molecules can act as enzymes (i.e.,
catalyze covalent changes in the structure of substrates, most of
which are also RNA molecules).
 Hence, many natural ribozymes catalyze either the hydrolysis of one

of their own phosphodiester bonds (self-cleaving ribozymes), or the
hydrolysis of bonds in other RNAs.
RIBOZYMES
 Some ribozymes have been found to catalyze the aminotransferase
activity of the ribosome.
 Examples of naturally occurring ribozymes include:

◦ Peptidyl transferase 23S rRNA
◦ RNase P
◦ Group I and Group II introns
◦ GIR1 branching ribozyme
◦ Leadzyme – initially created in vitro, natural forms have been found
◦ Hairpin ribozyme
◦ Hammerhead ribozyme
◦ HDV ribozyme
◦ Mammalian CPEB3 ribozyme
◦ VS ribozyme
◦ glmS ribozyme
◦ CoTC ribozyme
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Ribozymes – Hammerhead ribozyme
Structure of
Hammerhead
hammerhea
ribozyme (type I)
d ribozyme
Hammerhead
ribozyme (type III)
RIBOZYMES – Hairpin ribozyme
Secondary structure of a
minimal hairpin ribozyme with
substrate RNA bound. Circles
individual nts
A representation of the 3D structure

of the hairpin ribozyme
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EXAMPLES OF RIBOZYMES
 Ribonuclease P
◦ Almost all living things synthesize an enzyme
Ribonuclease P (RNase P) that cleaves the head (5') end
of the precursors of transfer RNA (tRNA) molecules.
◦ In bacteria, RNase P is a heterodimer containing a

molecule of RNA and one of protein.
◦ Separated from each other, the RNA retains its ability to

catalyze the cleavage step (although less efficiently than
the intact dimer), but the protein alone cannot do the
job.
Examples of ribozymes
 Spliceosomes
◦ Spliceosomes remove introns and splice the exons of most nuclear
genes. They are composed of 5 kinds of small nuclear RNA (snRNA)
molecules and a large number of protein molecules. It is the snRNA
— not the protein — that catalyzes the splicing reactions.
 The Ribosome is a Ribozyme
◦ Ribosomes are huge aggregates containing 3 (4 in eukaryotes) rRNA
molecules and scores of protein molecules. The three-dimensional
structure of the large (50S) subunit of a bacterial ribosome shows that
formation of the peptide bond that links each amino acid to the growing
polypeptide chain is catalyzed by the 23S RNA molecule in the large
subunit. The 31 proteins in the subunit probably provide the scaffolding
needed to maintain the three-dimensional structure of the RNA.
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APPLICATIONS OF
RIBOZYMES
 Some ribozymes may play an important role as therapeutic agents, as
enzymes which tailor defined RNA sequences, and for applications in
functional genomics and gene discovery. For example:
◦ Hairpin ribozymes have been modified in such a way that they can
be used to target cleavage of other RNA molecules.
◦ Hairpin ribozymes have been developed for potential therapeutic

use, e.g., preventing the replication of pathogenic viruses. Antiviral
hairpin ribozymes have been generated and expressed within
mammalian cells, and cells expressing different engineered
ribozymes have been shown to be resistant to infection by HIV-1,
hepatitis B, and Sindbis virus.
 Investigators studying the origin of life

have produced ribozymes in the
laboratory that are capable of
catalyzing their own synthesis under
very specific conditions, such as an
RNA polymerase ribozyme. What
evolutionary implications??
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Time lines…
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ANIMATIONS:
THE RNA WORLD HYPOTHESIS
AND RIBOZYMES
SOME PHYSICAL & CHEMICAL

PROPERTIES OF DNA and RNA
 Polarity and Electrophoretic ability.

 Solubility in water and solvents.
 Precipitation and effect of salt.
 Stability vs. pH ranges.
 Light Absorption spectra.
 DNA’s ability to be cut and re-joined –
Molecular scizors/bundages.
 Effect of Temperature.
 Ability to bind or be bound by proteins.
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Additional properties of DNA (and

RNA) and their usefulnes
1. Negatively charged, showing
polarity – able to migrate when
subjected to a potential difference
(voltage) – a technique reffered to as
electrophoresis.
Plant RNA Extraction: Cow peas leaves

DNA Electrophoresis
 The size of DNA/RNA fragment, medium/gel used, pH, and
voltage/current applied determines the migration (electrophoretic)
speed.
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GelDoc Documentation system

(BIO-RAD®)
Prof Arthur Tugume,

BOT2210/BBT1206
DNA Electrophoresis
Southern blotting and hybridization with probes can be used to locate a few specific
fragments in a large pool of DNA.
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Solubility of DNA and RNA
2. Solubility – both DNA and RNA are

soluble in aqueous solutions (e.g.,
water) at pH7.0 and room
temperature, but it is better to dissolve
them in the TE buffer at pH7.0-8.0.
However, at high concentrations
(≥10mg/ml, or ≥ 10μg/μl), dissolved
DNA/RNA is viscous). At lower concs,
one cannot detect the DNA/RNA by
sight or by noting the viscosity of the
solution. High temp. decrease
viscousity. DNA/RNA are insoluble in
organic solvents. DNA/RNA become
insoluble in the presence of
alchohol and salt.
Precipitation of DNA and RNA
3. Precipitation – a method used

to purify and/or concentrate
RNA, DNA (and polysaccharides
such as pectin and xyloglucan)
from aqueous solutions.
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More on precipitation of DNA & RNA

 Nucleic acids are precipitated by first ensuring that the correct
concentration of positive ions is present in solution (too much will
result in a lot of salt co-precipitating with nucleic acid, too little will
result in incomplete nucleic acid recovery) and then adding 2-3
volumes of at ≥95% ice-cold ethanol. Hence, ethanol precipitation is a
commonly used technique for concentrating and de-salting DNA or
RNA preparations in aqueous solution.
 The basic procedure is that salt and ethanol are added to the aqueous
solution, which forces the nucleic acid to precipitate out of
solution. The precipitated nucleic acid can then be separated from
the rest of the solution by centrifugation. The pellet is washed in cold
70% ethanol to remove excess salt then after a further centrifugation
step the ethanol is removed, and the nucleic acid pellet is allowed to
dry before being re-suspended/dissolved in clean aqueous buffer. SO
HOW DOES THIS WORK?

 Importance of solubility: First why nucleic acids are soluble in water?
Water is a polar molecule – it has a partial negative charge near the oxygen
atom due the unshared pairs of electrons, and partial positive charges near
the hydrogen atoms.
 Because of these charges, polar molecules, like DNA or RNA, can interact
electrostatically with the water molecules, allowing them to easily dissolve in
water. Polar molecules can therefore be described as hydrophilic and non-
polar molecules, which can’t easily interact with water molecules, are
hydrophobic. Nucleic acids are hydrophilic due to the negatively charged
phosphate groups along the sugar phosphate backbone.
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 Role of salt: The role of the salt in the protocol is to neutralize the charges
on the sugar phosphate backbone. A commonly used salt is sodium acetate. In
solution, sodium acetate breaks up into Na+ and [CH3COO]-. The positively
charged sodium ions neutralize the negative charge on the PO3- groups on
the nucleic acids, making the molecule far less hydrophilic, and therefore
much less soluble in water.
 Choice of salt:
 Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations.
 Use Sodium chloride (0.2M final conc) for DNA samples containing SDS since NaCl keeps SDS
soluble in 70% ethanol so it won’t precipitate with the DNA.
 Use Lithium Chloride (0.8M final conc) for RNA. This is because 2.5-3.0 volumes of ethanol
should be used for RNA precipitation and LiCl is more soluble in ethanol than is NaAc so will
not precipitate, but beware – chloride ions will inhibit protein synthesis and DNA polymerase
so LiCl is no good for RNA preps for in vitro translation or reverse transcription. In these cases,
use NaAc.
 Use Ammonium acetate (2M final conc) for the removal of dNTPs, but do not use for
preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the
enzyme.

 The role of the ethanol (or isopropanol): The electrostatic attraction
between the Na+ ions in solution and the PO3- ions are dictated by
Coulomb’s Law, which is affected by the dielectric constant of the solution.
Water has a high dielectric constant, which makes it fairly difficult
for the Na+ and PO3- to come together. Ethanol on the other hand has
a much lower dielectric constant, making it much easier for Na+ to interact
with the PO3-, shield it’s charge and make the nucleic acid less hydrophilic,
causing it to drop out of solution – Precipitation.
 Role of temperature: Incubation of the nucleic acid/salt/ethanol or

isopropanol mixture at low temperatures (e.g., -20 or -80C) is commonly
cited in protocols as necessary in protocols to enhance precipitation .
However, it may not always be a requirement because nucleic acids at
concentrations as low as 20ng/ml will precipitate at 0-4oC so incubation for
15-30 minutes on ice is sufficient.
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 To increase the yield in precipitations of low concentration or small

nucleic acid pieces (less than 100 nucleotides):
 Add MgCl2 to a final concentration of 0.01M.
 Increase incubation time on ice before centrifugation to 1 hour.
 Otherwise use kits – based on ion exchange chromatography columns.
Acidic and alkaline conditions
4. Acidic and alkaline conditions (pH) –

Infuence of pH on DNA and RNA? (Reading
assigment).
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The concept of Dilution factor

Primary unit of mass Equivalent 1. If an extract containing 914.72
ng/ml of DNA was diluted 79.7
times to allow downstream
Ton 1000kg applications, how much DNA (in
μg) will be present in 100μl of
Kilogram 1000g the dilution?
Gram 1000mg 2. Three samples of RNA extracts

from the same mammalian
tissues, named A, B, and C
Milligram 1000μg have RNA concentrations
753ng/μL, 259μg/mL, and
Microgram 1000ng 30ng/mL, respectively. If 100μl
of each RNA sample was used
Nanogram 1000pg to make a compound mixture of
300μL from the three RNA
Picogram 1000fg samples, calculate the
concentration in μg/μL of the
300μL RNA mixture
Note: 1mL = 1000μL, and 1L = 1000mL
Nucleic acid light absorption

spectra
5. Light absorption properties of DNA and RNA – The rings of
the bases are made up of alternating single and double bonds. Such
systems absorb in the UV, or if the system is large enough, in the
visible spectrum.
Each of the 4 nucleotide bases has a slightly
different absorption spectrum, and the
spectrum of DNA is the average of them.
A pure DNA solution appears transparent to
the eye, and absorption doesn't become
measurable until 320 nm. Moving further into
the UV region, there is a peak at about 260 nm,
followed by a dip between 220 and 230, and
then the solution becomes essentially opaque in
the far UV. A solution of double stranded, native
DNA, with a concentration of 0.04 mg/ml has an
absorbance (or optical density, OD) of about 1.0
at the 260 nm peak.
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Light abosrption of DNA and RNA
 The absorbance of DNA in the UV is the reason UV radiation can be used

to sterilize: the absorbed energy enhances destruction of the DNA and kills
the organism. It is also the reason UV causes skin cancer: the absorbed
radiation causes mutations which inactivate regulatory mechanisms needed
to control cell division.
When a DNA helix is denatured to

become single strands, e.g. by heating, the
absorbance is increased about 30%. This
increase, called the hyperchromic
effect, reveals the interaction between
the electronic dipoles in the stacked
bases of the native helix.
Light abosrption of DNA and RNA
 The absorbance (optical density, OD) of DNA/RNA is of key practical

application in quantitative spectrophotometric determination of the
nucleic acids; i.e., determination of DNA/RNA concentration, expressed
in units of mass per unit volume (e.g., mg/ml, μg/ml, or μg/μl).
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DNA isolated from

calf thymus
RNA isolated Light absorption

from cow peas maximum of DNA
and RNA is at 260nm
Spectrophotometric determination of
DNA, RNA and protein concentrations
 Using spectrophotometry to determine the concentration of nucleic
acids is based on the fact that there is a relationship between the amount
of light absorbed by a solution and the concentration of nucleic acids in
the solution.
 For many substances, the amount of light absorbed depends on the solute
concentration and the length of the light path; a relationship known as
Beer-Lambert law, expressed as: A=
ɛCL
where: A is the absorbance (or the optical density of the soln.
ɛ is a constant, the absorption coefficient (cm -3M-1)
C is the solute concetration (μg/ml)

L is the lengh of light path (cm)
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 A plot of absorbance versus concentration gives a straight line through
the origin with a slope equal to ɛ. Hence, a standard curve can be made
using DNA and RNA of known concentrations, and the standard curves
used to determine unknown concentrations of DNA and RNA isolations.
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 The integrated light path length, dilution factor and a wide array of data
analysis software allow for fast determination of [DNA] and [RNA] in
samples. Explain the concept of dilution factor!
 Light absorption at different wavelength is an intrinsic property of most

biological molecules, although most do not absorb light in the visible
range, but they do absorb ultraviolet light. As stated earlier, we can take
advantage of UV absorbance to quickly estimate the concentration and
purity of DNA, RNA, and proteins in a sample. Depending on the protein,
UV analysis of proteins at 280 nm has a linear range from about 0.1-5
mg/mL
 Proteins have two absorbance peaks in the UV region, one between 215-
230nm, where peptide bonds absorb, and another at about 280nm due
to light absorption by aromatic amino acids (tyrosine, tryptophan and
phenylalanine).
 As stated earlier for nucleic acid, purines have an absorbance
maximum slightly below 260 nm while pyrimidines have a
maximum slightly above 260 nm. Therefore, although it is common
to say that the absorbance peak of nucleic acids is 260 nm, in
reality, the absorbance maxima of different fragments of DNA vary
somewhat depending on their purine/pyrimidine composition.
 The absorptivity constant for a nucleic acid depends on its base

composition and on whether it is single-stranded or double-
stranded. Despite the fact that different proteins and nucleic acid
fragments vary in their absorptivity, analysts commonly use
“average” absorptivity constants to estimate the concentration of
nucleic acid or protein in a sample.
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 The average absorptivity constants for proteins and nucleic

acids lead to the following relationships:
 If a sample containing pure double-stranded DNA has an absorbance

of 1 at 260 nm, then it contains approximately 50 µg/mL of double -
stranded DNA.
 If a sample containing pure single-stranded DNA has an absorbance of
1 at 260 nm, then it contains approximately 33 µg/mL of DNA.
 If a sample containing pure RNA has an absorbance of 1 at 260 nm, then
it contains approximately 40 µg/mL of RNA.
 Values for proteins vary. A very rough rule is that if a sample containing
pure protein has an absorbance of 1 at 280 nm, then it contains
approximately 1 mg/mL of protein.
UV MEASUREMENTS OF DNA, RNA AND PROTEINS

WAVELENGTH SIGNIFICANCE COMMENTS
215-230 nm Minimum Measurements are generally not performed
absorbance for at this wavelength because commonly used
nucleic acids buffers and solvents, such as Tris, also absorb
peptide bonds in at these wavelengths.
proteins absorb
light
260 nm Nucleic acids have Purines absorbance maximum is slightly <260
maximum nm; pyrimidines maximum is slightly >260 nm.
absorbance Purines have a higher molar absorptivity than
pyrimidines. Therefore, the absorbance
maximum and absorptivity of a segment of
DNA depends on its base composition.
Proteins have little absorbance at 260 nm.
270 nm Phenol absorbs Phenol may be a contaminant in nucleic
strongly acid preparations.
280 nm Aromatic amino Nucleic acids also have some absorbance
acids absorb light at this wavelength.
320 nm Neither proteins nor Used for background correction since
nucleic acids neither nucleic acids or proteins absorb at
absorb this wavelength.
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Estimation of the purity of nucleic Acid

and protein preparations
 It is possible to use UV spectrophotometry to estimate the purity of a
solution of nucleic acids and proteins. This method involves measuring the
absorbance of the solution at two wavelengths, usually 260 nm and 280 nm,
and calculating the ratio of the two absorbances:
 An A260/A280 ratio of 2.0 is characteristic of pure RNA.

 An A260/A280 of 1.8 is characteristic of pure DNA.
 An A260/A280 ratio of about 0.6 is characteristic of pure protein
 Therefore, a ratio of 1.8-2.0 is desired when purifying nucleic acids. (Note

that this method does not actually distinguish DNA and RNA from one
another.) A ratio less than 1.7 means there is probably a contaminant in the
solution, typically either protein or phenol.
DNA Melting
6. The ability of DNA be denatured by heat treatment and re-
association (anealing) upon cooling – the concept of DNA meting.
◦ DNA Denaturation - also called DNA melting, is the process by
which double-stranded DNA unwinds and separates into single-
stranded strands through the breaking of hydrogen bonding between
the bases. May be heat denaturation or chemical denaturation (by
chemicals e.g., urea).
◦ DNA annealing – re-association by hydrogen bonds of two

separated complementary strands of DNA that were separated by
heat (thermally denatured).
◦ Denaturation and anealing of DNA strands have several applications in

DNA technology such as in PCR, hybridization assays, e.t.c.
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DNA Melting
 The process of DNA denaturation can be used to analyze some aspects of
DNA. Because CG base-pairing is generally stronger than AT base-pairing, the
amount of C and G in a genome (called the "GC content") can be estimated
by measuring the temperature at which the genomic DNA melts.
DNA Melting
 Therefore, the melting temperature (Tm) is defined as the temperature
at which half of the DNA strands are in the double-helical state and half are in
the random coil states. The Tm depends on both the length of the molecule,
and the specific nt sequence composition of that molecule. Molecules with
high GC and low AT content have high Tms and vice versa for high low GC
and AT content. Tm is also affected by pH and solution [salt].
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DNA AND RNA AS SUBSTRATES

FOR VARIOUS ENZYMES
 Ability to be copied, cut, joined, amended,

etc by various types of enzymes
◦ Polymerases
◦ Endonucleases/exonucleases
◦ Ligases
◦ Reverse Transcriptases
◦ Etc, etc
Prof Arthur Tugume,

BOT2210/BBT1206
Activity of restriction endonucleases and

DNA Ligase enzymes on DNA
7. The ability of DNA fragments to be cut and re-joined – the function of
restriction endonucleases and DNA ligase, respectively.
◦ A restriction enzyme (or restriction endonuclease) is an enzyme that cuts
double-stranded DNA at specific recognition nucleotide sequences known
as restriction sites.
◦ Such enzymes, found in bacteria and archaea, are thought to have evolved
to provide a defense mechanism against invading viruses. Inside a
bacterial host, the restriction enzymes selectively cut up foreign DNA in a
process called restriction; host DNA is methylated by a modification
enzyme (a methylase) to protect it from the restriction enzyme’s activity.
◦ To cut the DNA, a restriction enzyme makes two incisions, once through
each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
◦ Over 3000 restriction enzymes have been studied in detail, and >600 of
these are available commercially and are routinely used for DNA
modification and manipulation in laboratories as the most important tools in
recombinant DNA technology tool box.
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Restriction endonucleases
 Restriction enzyme recognition site – a specific sequence of nucleotides
recognized and cut by the restriction enzyme produce a double-stranded cut in
the DNA. While recognition sequences vary between 4 and 8 nucleotides,
many of them are palindromic (which correspond to nitrogenous base
sequences that read the same backwards and forwards).
EXAMPLES
Recognition site for restriction Recognition site for restriction

enzyme EcoRI, which produces enzyme SmaI which produces
"sticky" ends "blunt" ends
 Restriction enzyme nomenclature – Since their discovery in the 1970s,
more than 100 different restriction enzymes have been identified in different
bacteria. Each enzyme is named after the bacterium from which it was isolated
using a naming system based on bacterial genus, species, and strain. For
example, the name of the EcoRI restriction enzyme was derived as shown
below.
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 Restriction enzyme examples – >3000 characterized today!
Enzyme Source Recorgnition site Cut
EcoRI Escherichia coli 5'GAATTC 5'---G AATTC---3‘

3'CTTAAG 3'---CTTAA G---5'
EcoRII Escherichia coli 5'CCWGG 5'--- CCWGG---3‘
3'GGWCC 3'---GGWCC ---5’
EcoRV Escherichia coli 5'GATATC 5'---GAT ATC---3'
3'CTATAG 3'---CTA TAG---5'
BamHI Bacillus 5'GGATCC 5'---G GATCC---3‘
amyloliquefaciens 3'CCTAGG 3'---CCTAG G---5'
PovII Proteus vulgaris 5'CAGCTG 5'---CAG CTG---3‘
3'GTCGAC 3'---GTC GAC---5'
XbaI Xanthomonas 5'TCTAGA 5'---T CTAGA---3'
badrii 3'AGATCT 3'---AGATC T---5'
StuI Streptomyces 5'AGGCCT 5'---AGG CCT---3'
tubercidicus 3'TCCGGA 3'---TCC GGA---5'
PstI Providencia 5'CTGCAG 5'---CTGCA G---3'
stuartii 3'GACGTC 3'---G ACGTC---5'
DNA Ligase
 DNA Ligase – a specific type of enzyme, that repairs single-stranded
discontinuities in double stranded DNA molecules. It is able to join both
sticky and blunt ends of DNA.
 The mechanism of DNA ligase is to form two covalent phosphodiester

bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the
5' phosphate end of another ("donor").
32
3/26/2024
DNA can be bound by proteins

8. Proteins binding of DNA - DNA-binding proteins are proteins that are
composed of DNA-binding domains and thus have a specific or general
affinity for either single or double stranded DNA. Sequence-specific DNA-
binding proteins generally interact with the major groove of B-DNA,
because it exposes more functional groups that identify a base pair.
However there are some known minor groove DNA-binding proteins.
Within chromosomes, DNA is held in complexes with structural proteins.

These proteins organize the DNA into a compact structure called
chromatin. In eukaryotes this structure involves DNA binding to a
complex of small basic proteins called histones, while in prokaryotes
multiple types of proteins are involved. The histones form a disk-shaped
complex called a nucleosome, which contains two complete turns of
double-stranded DNA wrapped around its surface. These non-specific
interactions are formed through basic residues in the histones making
ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are
therefore largely independent of the base sequence.
Nucleosome: Interaction of DNA

(shown in orange) with histones
(shown in blue). These proteins' basic
amino acids bind to the acidic The lambda repressor helix-turn-
phosphate groups on DNA. helix Transcription factor bound to
its DNA target
33
3/26/2024

 Chemical modifications of these basic amino acid residues include
methylation, phosphorylation and acetylation. These chemical changes
alter the strength of the interaction between the DNA and the
histones, making the DNA more or less accessible to transcription
factors and changing the rate of transcription.
 Other non-specific DNA-binding proteins in chromatin include the
high-mobility group proteins, which bind to bent or distorted DNA.
These proteins are important in bending arrays of nucleosomes and
arranging them into the larger structures that make up chromosomes.
 A distinct group of DNA-binding proteins are the DNA-binding
proteins that specifically bind single-stranded DNA. In humans,
replication protein A is used in processes where the double helix is
separated, including DNA replication, recombination and DNA repair.
These binding proteins seem to stabilize single-stranded DNA and
protect it from forming stem-loops or being degraded by nucleases.

 Restriction enzymes differ from gene regulatory proteins being much more
fastidious (particular) in their substrate choice (a single base pair change
eliminates recognition by the protein) and having shorter recognition sequences
(4-8 bp vs. 14-20 bp) with strict symmetries. With molecules such as histones or
E. coli trp repressor (regulatory proteins) local conformation and configuration of
DNA appears to be the only form of recognition.
 In contrast, other proteins have evolved

to bind to particular DNA sequences,
notably the various transcription
factors, which are proteins that
regulate transcription. Each
transcription factor binds to one
particular set of DNA sequences and
activates or inhibits the transcription of
genes that have these sequences close
to their promoters. The restriction enzyme EcoRV (green) in a
complex with its substrate DNA
34

RNA World Hypothesis, Properties of DNA&RNA

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3/26/2024

BBT1206: STRUCTURE AND

BOT2210: MOLECULAR STRUCTURE

The RNA World Hypothesis & Ribozymes;

Prof. Arthur K.Tugume (B.Sc., M.Sc., PhD)

Department of Plant Sciences, Microbiology & Biotechnology,

TOPICAL COURSE OUTLINE

Prof Arthur Tugume,

Prof Arthur Tugume, BOT2210/BBT1206

Prof Arthur Tugume,

Prof Arthur Tugume,

Prof Arthur Tugume,

WHAT IS THE RNA WORLD

RNA WORLD & THE ORIGIN OF

◦ DNA encodes the genetic information of proteins but DNA replication

◦ But if RNA can serve both as a repository of information (in its

RNA world & the origin of life

 RNA is able both to store genetic information, like DNA, and to

 Thomas Čech (2011) has argued that that multiple self-replicating

Prof Arthur Tugume, BOT2210/BBT1206

RNA world & the origin of life

 DNA is thought to have taken over the role of data storage

Prof Arthur Tugume, BOT2210/BBT1206

IMPORTANT ENZYMATIC PROPERTIES OF

 RNA enzymes, or ribozymes, are still found in today's DNA-based life

◦ The ability to self-duplicate, or duplicate other RNA molecules.

◦ The ability to catalyze simple chemical reactions which would

Prof Arthur Tugume, BOT2210/BBT1206

IMPORTANT ENZYMATIC PROPERTIES OF

◦ The ability to catalyze the formation of peptide bonds, in

Prof Arthur Tugume, BOT2210/BBT1206

WHY SHOULD A CELL STORE GENETIC

Prof Arthur Tugume, BOT2210/BBT1206

Adenine (A) Guanine (G)

Cytosine (C) Thymine (T) Uracil (U)

Prof Arthur Tugume, BOT2210/BBT1206

WHY SHOULD A CELL STORE GENETIC

 The chemical properties of RNA make large RNA molecules inherently

 These limitations do not make use of RNA as an information storage

 Hence, many natural ribozymes catalyze either the hydrolysis of one

Prof Arthur Tugume, BOT2210/BBT1206

 Examples of naturally occurring ribozymes include:

Ribozymes – Hammerhead ribozyme

Prof Arthur Tugume, BOT2210/BBT1206

RIBOZYMES – Hairpin ribozyme

A representation of the 3D structure

◦ In bacteria, RNase P is a heterodimer containing a

◦ Separated from each other, the RNA retains its ability to

Prof Arthur Tugume, BOT2210/BBT1206

Prof Arthur Tugume, BOT2210/BBT1206

◦ Hairpin ribozymes have been developed for potential therapeutic

Prof Arthur Tugume, BOT2210/BBT1206

 Investigators studying the origin of life

Prof Arthur Tugume, BOT2210/BBT1206

Prof Arthur Tugume, BOT2210/BBT1206

Prof Arthur Tugume, BOT2210/BBT1206

Prof Arthur Tugume, BOT2210/BBT1206

SOME PHYSICAL & CHEMICAL

 Polarity and Electrophoretic ability.

Additional properties of DNA (and

Plant RNA Extraction: Cow peas leaves

Prof Arthur Tugume, BOT2210/BBT1206

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