MEDS2003: BIOCHEMISTRY AND MOLECULAR BIOLOGY

LECTURE ONE: INTRODUCTION TO MOLECULAR BIOLOGY

DNA and Information technology (IT)

Information must be able to:

- Stored (stable, corruption-free, protected, backed up)

- Accessed (easily)
- Retrieved (quickly)
- Copied (accurately)
- Read (de-coded easily and selectively)
- Used (appropriately)

Information Storage

Information must be able to be stored (in cells):

- Stable, resistant to attack by all the compounds inside the cell, e.g., free radicals’ oxygen as
oxygenation is a big problem in cells
- Corruption-free meaning the information cannot be modified – information is stored inside the
double helix where it is protected – many backups of genome and each copy is double helixes
- Protected against unwanted access e.g., viral sequences
- Backed up so it can be repaired

Silicon IT Comparison DNA

- RAM – mRNA
- Hard Drive – DNA
- Printer - Proteins /
rRNA tRNA
- USB sticks – genetic
information passing
through
Information management in the body

- Short term memory: The transient copy of some of the information about events of the day (mRNA)
- Long term memory, all the information contained in the brain as stable copy (DNA)
- What the brain controls, The organs and the body actions, doing molecules (proteins / rna)

Carbon Based IT

DNA  transcription  RNA  translation  Protein

DNA  replication  DNA
RNA  Reverse transcription  DNA

- We have a lot more control in gene expression in Eukaryotes

- The genome (DNA)

- The transcriptome (the RNA)
- The proteome (the protein)

The transcriptome

There are a few types of RNA present within the body

- Micro RNA - small single-stranded non-coding RNA molecule (containing about 22 nucleotides) found

in plants, animals, and some viruses, that functions in RNA silencing and post-transcriptional
regulation of gene expression.
- Small nuclear RNA – Small nuclear RNAs (snRNAs) are critical components of the spliceosome that
catalyse the splicing of pre-mRNA.
- Transfer RNA (approximately 15% by weight) – adapter molecule
- Messenger RNA (1-2% by weight) – encoding genes
- Ribosomal RNA (80% by weight) – protein factory
Proteome

Messenger RNA  Protein (folding occurs). Number of proteins in the body

- Ion channels – metabolism, control uptakes into the cell and things out of the cell
- Receptors – important for binding (drugs)
- Antibodies – immunological response
- Enzymes – biological catalyst that speed up rate of reactions
- Transcription factors – switch genes on/off – controls gene expression

The Central Dogma

- Describes the flow of genetic information

DNA  transcription  RNA  translation  Protein

DNA  replication  DNA
RNA  Reverse transcription  DNA
RNA to RNA can happen in some viruses
 - Reverse translation is not possible
- Cannot skip from DNA to protein without RNA
- Cannot make DNA from protein

Crick’s Central Dogma Prediction

- He predicted DNA can go to proteins straight away which was wrong

- As RNA is ancient  everything always goes via RNA
- “Crick gave his lecture - "On protein synthesis" - at University College London for the Society for
Experimental Biology. …Crick delivered four predictions about genes - and their link to the proteins
that build our bodies. In each of these ideas, he was right” Matthew Cobb”
Information Management

- The DNA in almost every cell in your body contains the same information – etc heart cells & muscle
cells – just needs genes to become the different types of the cell – The DNA is still the same
o A complete set of genes to code for every protein
o There is usually only one copy of each gene on a chromosome.
- Each cell will need some proteins in massive numbers, many at low numbers and some not at all
- Transcriptome & Proteome can be different

The Discovery of Nucleic Acid

- First isolated by a Swiss Biochemist Fredrich Miescher circa 1860 from pus on bandages.
- The new polymer contained the usual suspects C, O, H and N and also Phosphorus (this is not in
protein)

Extra big molecule (phosphorus)  everyone originally though that the proteins contained all the information
BUT phosphorus was not a protein.

History

- It was named nucleic acid because it was first isolated in the nucleus, and it was acidic
- It was not thought to be the genetic material. That role was thought to be fulfilled by proteins as they
contained more potential for variation (20 different amino acids rather than 4 different nucleotides).
o Because 20 was more than 4, they thought the proteins contained genetic information.

The history of nucleic acids

- Griffith’s transforming principle

- Avery, McCarty, and MacLeod
- Hershey Chase and the Waring blender
- Chargaff’s rules
- Watson and Crick

These researchers all contributed massively to our understanding of the molecular basis of inheritance.

The role of DNA as genetic material

Work by Griffith then

MacLeod, McCarty, and Avery.
Using Pneumococcus, they
were able to transform the
bacterium from the rough (R)
non-pathogenic form to the
smooth (S) pathogenic form by
adding a killed lysate of S
bacteria. By a process of
elimination, the active
ingredient in the killed S
bacterial lysate was identified
as DNA.
The role of DNA as genetic material

This was further confirmed by the Waring Blender experiment (Hershey and Chase).

- The bacteriophage T2 which infects certain bacteria injects some genetic material…. BUT WHAT IS IT?
- The phage was labelled by growing it in – 35S (which labels protein only  methionine  contain
sulphur) or – 32P (which only labels DNA).

- Bacteriophage T2 (a virus that infects certain bacteria) injects genetic information into its victims –
But what is it?
- Phage was labeled with 35S (which labels protein only) and 32P (which only labels DNA).
- During infection, the phage was knocked off the bacteria with the blender….
 - The 35S was found in the supernatant, not the pellet. Hence the protein part of the phage was not
transferred to the bacteria.
- The 32P was found in the pellet, indicating the DNA was transferred to the bacteria.

The Role of DNA as genetic material

- So nucleic acid really was the genetic information

- Suddenly the ‘boring’ polymer is the subject of intense research
- How the chemistry and structure of the polymer enables it to store information

The Chemistry of DNA

- Chargaff’s Rules:
• The #sugars (deoxyribose) = # phosphates = # bases
• The amount of purine = pyrimidine.
• A = T; G = C

The Birth of the Double Helix

- Photograph taken by Rosalind Franklin

Watson and Crick

Lecture 1 questions

Which of the following never happens in any cell?

A. the synthesis of RNA from a DNA template

B. the synthesis of DNA from a DNA template
C. the synthesis of DNA from an RNA template
D. The synthesis of RNA from an RNA template
E. the synthesis of DNA from a protein template – there is no reverse translation so isn’t possible

True or False: The bases in DNA and RNA are joined by phosphodiester bonds

LECTURE TWO: NUCLEIC ACID STRUCTURE

The Bases

- The pictures show the most common form at

physiological PH
o They can take different forms
depending on pH
o Low pH -> high hydrogen
o High pH -> low hydrogen
- Purines: Adenine and Guanine
- Pyrimidines: Cytosine, Thymine and Uracil
Chargaff’s Rules

- Erwin Chargaff was able to purify purines and pyrimidines and quantify them from different
organisms.

The bases

- There is hydrogen boding happening between the bases

- Green is H donor
- Red is H bond acceptor  then they pair up and form hydrogen bond

- Adenine as one acceptor with matches with Thymine and Uracils one donor
 - 3 hydrogen bonds  so CG stronger than AT

What might lead to changes in protonation states?

- Protonation  more H+
- Deprotonation  less H+
- If it is too basic it can deprotonate
- This can lead to changes in hydrogen bonding capability

Sugar

1’: N-glycosidic (nitrogen from the base attaching to the sugar) bond attaches here
2’: OH for ribose, H for deoxyribose
3’: hydroxyl (OH)  important in replication as nucleotides gets added here
5’: phosphate attaches here

Sugar-Phosphate Backbone
The Double Helix

What is holding the double helix together?

- Hydrogen bonds
- Ionic Interactions
- Van der Waals interactions
- Hydrophobic interactions

How do we disrupt these?

Hydrogen Bonds

● Hydrogen bonding between bases gives double helix its

specificity.
● A with T: amine group on A acts as a donor to the keto group of T
and the ring N of A acts as an acceptor, the ring N of T (or U in RNA)
is protonated at physiological ph.
● G with C: amine groups of C and G act as donors & the keto
groups as acceptors

- Hard to open DNA if there its lots of GC

Ionic Interactions

- Backbone is full of negatively charged phosphates

- Repulsive
- Other interactions favour base pairing
- Result: Twist
● Forces give DNA its twist - Phosphate group in each residue is negatively charged at physiological pH causing
the two phosphates to repel away from each other. ● Bases actually want to associate together but the
phosphates want to be as far away as possible. ● The net result is the twist to accommodate both of the forces
above.

Base Stacking

- Bases have a ring structure  aromatic

- Hydrophobic and wants to stay away
from the water
- ● Bases are flat & aromatic - thus,
hydrophobic (hydrophobic molecules are
hidden in the middle) ● Bases are buried in
the interior of the molecule as the bases
are flat meaning they can stack
favourably on top of each other at a
distance of 3.4A. ● Mobile electrons of
the aromatic rings (electron clouds) gain a
favourable interaction when in co
- Leads to attraction via van der Waals interactions

Bases All Absorb at 260 nm

At 260nm:

[DNA] of 50 ng/ul gives absorbance of 1

[RNA] of 40ng/ul gives absorbance of 1

Why does RNA absorb slightly more?

- Due to base stacking, double

stranded nucleic acid absorb
less at 260 nm
Major and Minor Grooves

- Binding of Zinc finger transcription factor to the major groove  specificity  needs information
- Binding of DAPI (DNA dye) to the minor groove  because it doesn’t have specificity
- Grooves on the surface gives protein access to the base pairs
o Grooves are essential to read the sequence.
o Proteins mainly access the major groove.
- Major grooves are the window into the base sequence and the information, “the soul of the DNA”.
- Many drugs will bind to the major or minor groove.
Interactions with Major Groove

Storing Information in DNA

- Information (hydrophobic bases) is buried by hydrophilic backbones of sugars and negative

phosphates  hard to access hydrophilic  hydrophobic (negatively charged phosphate) 
hydrogen bond  needs to be very specific if access is required
- DNA is double stranded – backup  replication

DNA vs RNA for Long Term Storage

RNA degrades easily because of ribonuclease  therefore it is not as stable

You can run a gel - if it’s degraded it will appear smeared - ribonuclease contamination
 - RNA has hydroxyl group attached to the sugar
- Attaching the hydroxyl group makes it unstable as it can be deprotonated in high PH (too basic
environment)
- This addition makes it negative and then looks for things that are positive (phosphate) and attacks
that
- This then breaks the bond into two derivatives
- DNA is safe from this attack as no hydroxyl is attached

RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is
broken, cleaving the RNA molecule. RNA is susceptible to this base-catalyzed hydrolysis because
the ribose sugar in RNA has a hydroxyl group at the 2’ position.[1] This feature makes RNA chemically unstable
compared to DNA, which does not have this 2’ -OH group and thus is not susceptible to base-catalyzed
hydrolysis

Deamination of Cytosine

- Gets deaminated as NH2 becomes oxygen  used for detection  this is where backup comes in as it
happens a lot
- Estimated to occur spontaneously 1100-500 times per cell, per day
 - Corruption in the code
- Uracil is recognised and removed from DNA by base excision repair

LECTURE 3: PROKARYOTIC REPLICATION

Meselson-Stahl Experiment (1958)

- DNA replication is semi-conservative.

- Started with their heavier template. They have a medium template that they add it to.
- From generation 0 to 1 the parent dna is still there just in small amounts

Copying DNA in a Nutshell (cell or tube)

- 1. Separate DNA strands

- 2. Add primers
- 3. Complementary base H-bonds – polymerase forms the phosphodiester bonds
- 4. Nucleotide added to strand
- 5. Optional: Proofread – not optional for replication but for PCR its not needed
- 6. Repeat steps 3 to 5… All the way to the end

Escherichia coli (E. coli)

- One circular chromosome of 4.6 million base pairs

- Needs to copy all of it during replication Does this in less than 40 min*
 - All this needs to be fast and accurate

1. Separate DNA strands

DnaA • Recognises oriC site – the origin of replication • Separates DNA at AT-rich region Helicase
(DnaB) • Loaded to each strand • Moves 5’ to 3’, unwinding the DNA Single-stranded-DNA-binding
protein (SSB) • Keeps DNA separated