Learning goals Case 2: “the most beautiful experiment in biology”

1. Structure of DNA and RNA.

DNA:
DNA is made up of molecules called nucleotides. Each nucleotide contains a phosphate
group, a sugar group and a nitrogen base. The four types of nitrogen bases are adenine (A),
thymine (T), guanine (G) and cytosine (C). The sugar group is deoxyribose.
The primary structure of DNA is as follows: you do have sugar group, phosphate group, sugar
group, phosphate group attached in line. To the sugar groups the nitrogen bases are
attached. If you have two of these strands you can form an α-helix where A is always a bond
with T and G always a bond with C. The AT-connection contains 2 hydrogen bonds and the
GC-connection 3 hydrogen bonds. The AT-connection is 1,11 nm long and the GC-connection
is 1,08 nm long. Because of this and both hydrophilic and hydrophobic interactions between
the molecules the DNA is twisted into the so called α-helix or placed in a -sheet. These
combined will form the tertiary structure.
Adenine and guanine are purines. Thymine, cytosine and uracil are pyrimidines.
RNA:
RNA is made up of molecules called nucleotides. Each nucleotide contains a phosphate
group, a sugar group and a nitrogen base. The four types of nitrogen bases are adenine (A),
uracil (U), guanine (G) and cytosine (C). The sugar group is ribose.
The primary structure of RNA is as follows: you do have sugar group, phosphate group, sugar
group, phosphate group attached in line. To the sugar groups the nitrogen bases are
attached. RNA is always single stranded.

Nucleosomes -> a structural unit of a eukaryotic chromosome, consisting of a length of DNA

coiled around a core of histones.
Chromatins -> a substance within a chromosome consisting of DNA and protein. The DNA
carries the cell's genetic instructions. The major proteins in chromatin are histones, which
help package the DNA in a compact form that fits in the cell nucleus. Lower in condensation
order than chromosomes.

loops ->
They wrap around themselves to form supercoiled structure
Chromatid -> two chromatids form a chromosome
Centromere -> place where two chromatids are attached
Telomere -> A telomere consists of repeating pieces of DNA that protect the important
genes from shortening the chromosomes. The telomere is also important for the stability of
the chromosome.

Structure of the nitrogen bases

 2. Which are the differences between DNA and RNA?

DNA RNA

Full Name Deoxyribonucleic acid Ribonucleic acid

Replicates and stores genetic Carry out instructions encoded

Function
information in DNA

Structure Two strands One strand

Deoxyribose (which has one less

Sugar Ribose
hydroxyl group than ribose)

Adenine + Thymine Adenine + Uracil

Base Pairs
Guanine + Cytosine Guanine + Cytosine

Pairs Bonded
Hydrogen bonds Hydrogen bonds
By

Location in Form in nucleolus, then move

Mostly nucleus, some in mitochondria
Cells to cytoplasm

Length Several million base pairs Several thousand base pairs

Replication Self-replicating Synthesized by transcription

Reactivity Fairly stable More reactive

3. How is DNA formed (replicated)? https://www.mechanobio.info/genome-

regulation/how-is-dna-replicated/

Initiation
The stage for DNA replication is set in the G1 phase of the cell cycle and DNA is synthesized in the
S phase. DNA replication is initiated at specific sites in the genome known as the ‘origins’ which
are recognized and bound by origin binding proteins. Replication commences at a single origin in
prokaryotes and at multiple origins in eukaryotes, however, the basic mechanism of replication is
conserved in all organisms.
In eukaryotes, initiator proteins recruit the replicative helicase. The eukaryotic replicative helicase
is a complex of proteins called the CMG helicase. This assembly of the pre-replicative complex
(pre-RC) at origins during G1 phase is called ‘origin licensing’ FIG. The helicase is inactive in the
pre-RC and is activated only in the S phase when origins ‘fire’. Once origins fire, DNA synthesis
begins, and the initiator proteins are degraded or exported out of the nucleus to prevent re-
replication. The precise mechanisms of origin licensing and origin firing in two separate phases of
the cell-cycle ensure that DNA replication occurs only once per cell-cycle.

Synthesis
The mechanism of DNA replication is greatly influenced by DNA structure. The complementary
base pairing between the nitrogen bases A-T and G-C underlies the semi-conservative nature of
DNA replication, which results in a duplicated genome with one parental strand and one newly
synthesized strand. Each strand serves as a template for the DNA polymerase to catalyse the
addition of the correct base during synthesis of a new complementary strand. As the strands are
antiparallel with opposing polarity and since DNA polymerases can only synthesize DNA in the 5′
to 3′ direction, only one strand is continuously synthesized. This strand is called the leading
strand. Synthesis of the other strand, called the lagging strand, is made possible through
discontinuous synthesis of short fragments, called Okazaki fragments, in the 5′ to 3′ direction,
which are later joined together.
DNA synthesis begins in S phase as the replicative helicase unwinds and separates the two
strands of the DNA double helix. DNA polymerases ‘read’ the template strand and add the correct
complimentary base. DNA polymerases cannot synthesize DNA de novo and require a pre-
existing primer with a free hydroxyl group to add nucleotides and extend the chain. A specialized
RNA polymerase called primase synthesizes short RNA sequences about 10 nucleotides long
which serve as primers. A single primer aids DNA replication on the leading strand and multiple
primers initiate Okazaki fragment synthesis on the lagging strand. In Eukaryotes, the primase is
part of the DNA polymerase α. The replicative helicase and primase functionally co-operate and
stimulate each other’s activity.
After DNA polymerase α has synthesized a short, 30-40 nucleotide stretch of DNA, further DNA
synthesis is handed over to polymerase ε and polymerase δ which have a higher processivity
than polymerase α. The polymerase switching enables DNA synthesis with high fidelity as
polymerase ε and polymerase δ have a 3′ – 5′ exonuclease activity which enables proof reading
and removal of any incorrect bases that is incorporated. The Okazaki fragments which are about
100-200 nt in eukaryotes are ligated together in a process known as Okazaki fragment maturation
to complete DNA synthesis. Polymerase δ, as it runs into the adjacent Okazaki fragment ahead of
polymerization removes 2 to 3 nucleotides of the RNA primer thereby generating a short flap that
is processed by Fen1. This leaves a nick that is sealed by DNA ligase1.

5’ and 3’ https://www.youtube.com/watch?v=IV53GZGr11g

- Pol α is responsible for the initiation of DNA replication at origins of replication (on
both the leading and lagging strands) and during synthesis of Okazaki fragments on
the lagging strand.
- Pol δ is the DNA synthetic workhorse in the eukaryotic cell. Besides its essential
function on the lagging strand of the DNA replication fork, this enzyme also
functions during DNA recombinational processes, various DNA repair processes, and
even during damage-induced mutagenesis in the cell.
- Pol ε is required for genome duplication and tumor suppression. It supports both
replisome assembly and leading strand synthesis; however, the underlying
mechanisms remain to be elucidated.
- Pol ε and Pol δ are the only two eukaryotic nuclear DNA polymerases with an intrinsic
3'–5' exonucleolytic activity with which to excise primer terminal nucleotides (10).
This activity is especially useful for proofreading of errors made by these
polymerases.
- DNA-polymerase I: fills gaps left by removal of RNA primer (5’-3’exonuclease activity)
and involved in DNA repair
- DNA-polymerase II: involved in DNA repair
- DNA-polymerase III: chromosome-replicating enzyme
- Although non-coding DNA and RNA strands do not directly code for protein to be
made, they often serve to regulate which genes are made into protein in many cases.
4. How is RNA formed?
Using the DNA strand as template, a long chain of nucleotides is formed. This is called
transcription. Initiation of transcription begins with the binding of the enzyme to a promoter
sequence in the DNA. This region controls the reading of the DNA and formation of the RNA
strand.
The DNA is a double helix and two strands are wound tightly and the whole thing is twisted
over itself. As a first step the DNA double helix is unwound by the helicase activity of the
enzyme.
The DNA strand is then read from the 3’ to 5’ direction and a complementary RNA is formed
with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where
termination of RNA synthesis will occur.
Promoter: Promoter sequences are DNA sequences that define where transcription of a
gene by RNA polymerase begins. Promoter sequences define the direction of transcription
and indicate which DNA strand will be transcribed; this strand is known as the sense strand.
Enhancers: Enhancers are short regulatory elements of accessible DNA that help establish
the transcriptional program of cells by increasing transcription of target genes. They are
bound by transcription factors, co-regulators, and RNA polymerase II.
Transcription factors: Transcription factors are proteins involved in the process of
converting, or transcribing, DNA into RNA. One distinct feature of transcription factors is that
they have DNA-binding domains that give them the ability to bind to specific sequences of
DNA called enhancer or promoter sequences.

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Differences prokaryotes and eukaryotes.

-Prokaryotic DNA: Prokaryotic DNA is found in the cytoplasm of prokaryotic cells as well as
circular plasmids.
Eukaryotic DNA: Eukaryotic DNA is found in the nucleus of the cell, inside the chloroplast and
mitochondria.

-Prokaryotic DNA: Prokaryotic DNA is not found inside organelles.

Eukaryotic DNA: Some of the eukaryotic DNA is found inside chloroplast and mitochondria as
well.

-Prokaryotic DNA: The size of the DNA is less than 0.1 pg in prokaryotes.
Eukaryotic DNA: The size of the DNA is high in eukaryotes, usually more than 1 pg.

-Prokaryotic DNA: GC content is more than the AT content.

Eukaryotic DNA: AT content is more than 4xGC content.

-Prokaryotic DNA: Prokaryotic DNA consists of one copy of the genome.

Eukaryotic DNA: Eukaryotic DNA consists of more than one copies of the genome.

-Prokaryotic DNA: Prokaryotic DNA contains a small number of genes.

Eukaryotic DNA: Eukaryotic DNA contains a large number of genes.

-Prokaryotic DNA: Prokaryotic DNA is organized into a single chromosome.

Eukaryotic DNA: Eukaryotic DNA is organized into many chromosomes.
-Prokaryotic DNA: Prokaryotic DNA is not packed with histones.
Eukaryotic DNA: Eukaryotic DNA found in the nucleus packed with histones.

-Prokaryotic DNA: Prokaryotic DNA is circular. Hence, they do not have ends.
Eukaryotic DNA: Eukaryotic DNA is linear, containing two ends.

-Prokaryotic DNA: Introns are absent in prokaryotic DNA.

Eukaryotic DNA: Eukaryotic DNA consist of introns, interrupting the sequence of the coding
region.

-Prokaryotic DNA: Prokaryotic DNA contains fewer amounts of non-functional DNA.

Eukaryotic DNA: Eukaryotic DNA contains higher amounts of non-functional DNA.

-Prokaryotic DNA: Prokaryotic DNA lacks transposons.

Eukaryotic DNA: Eukaryotic DNA consists of transposons.

-Prokaryotic DNA: Prokaryotic DNA replication occurs in the cytoplasm.

Eukaryotic DNA: Eukaryotic DNA replication occurs in the nucleus.

-Prokaryotic DNA: Prokaryotic chromosome contains a single origin of replication.

Eukaryotic DNA: Eukaryotic chromosome contains many origins of replication.

-Prokaryotic DNA: Prokaryotic DNA replication is rapid; 2000 nucleotides are added per
second.
Eukaryotic DNA: Eukaryotic DNA replication is slow; 100 nucleotides are added per second.

INITIATION, ELONGATION, TERMINATION

https://www.youtube.com/watch?v=TNKWgcFPHqw
Helicase opens the a-helix
Primase (special RNA-polymerase) makes the primers
DNA-polyemerase makes the new strands to the existing strands
Exonuclease removes the primers between the Okazaki fragments
DNA-ligase seals op the newly made fragments
!This process is semiconservative!