BIOC2001 LECTURE NOTES

Lecture 1: Nucleic Acids

DNA -> made up of 1 sugar (deoxyribose) + 1 base (can be Purine: Adenine (A)/ Guanine (G) or
Pyrimidine: Cytosine (C)/ thymine (T)) + 3 phosphate groups (α, β, ¥).

Nucleoside = base + sugar

Nucleotides = base + sugar + phosphate

Chargaff’s rule:

In DNA the number of: A=T; G=C

Each one form a base pair. REMENBER!! G=C is joined by 3 H-bonds, meanwhile A=T only 2. This
difference in H bonds give DNA flexibility for certain processes.

Base pairing interactions provide specificity through precise requirements for H bonding.

The orientation of the 2 strands is antiparallel which means that 5’  3’ directions are opposite.

Enzymes that act on DNA

Nucleases cleave phosphodiester bonds. They can be endonuclease (cleaves bonds internally) or
exonucleases (start at one end and cleaves bonds one at time).

Restriction endonucleases (type 2): cleave both strands of DNA at specific sequences. Cuts can be
staggered. Recognition sites are palindromic (they read the same forward and backward).
Examples:

EcoR1: Escherichia coli R1 recognises 5’ –G’AATTC- 3’ and cut DNA forming sticky ends.

Pst1: Pseudomonas stuartii 1 recognises 5’-CTGCA’G -3’ and also forms sticky ends.

Hpa1: Haemophilus parainfluenzae 1 recognises 5’-GTT’AAC-3’ cuts DNA forming blunt ends.

DNA ligases: catalyse the formation of phosphodiester bonds between the 5’ -phosphate and 3’ –OH
of adjoining nucleotides in duplex DNA

Check mechanism in slides

DNA strands can separated by enzymes or in lab conditions (applying heat, high pH).

DNA topology:

There are some enzymes that can remove supercoiling such as:

Topoisomerase type I (in vivo): introduce transient single-stranded or double-stranded breaks into
DNA.

Restriction endonucleases (in lab): linearise plasmid DNA

Lecture 2: Plasmids

Features of plasmids:

 It is an extrachromosomal DNA molecule.

 Can be circular o linear
 They have an autonomous replication – don’t depend on host
 Range in size from Kbases to Mbases.
 Can control their copy number
 Partitioning: ensure inheritance at each cell division
 Incompatibility: plasmids with the same replication mechanism cannot co- exist in
the same cell. Several plasmids can co-exist in the same cell but not all can co-exist
stably. Incompatible plasmids are in the same incompatibly (Inc) group.
 Host range: can be of 2 types:
- Narrow-host-range: can only replicate in related species. Example: E.coli
plasmids can replicate in other E.coli cells.
- Broad-host-range: plasmids can replicate in a variety of hosts: RP4 can replicate
on Gram positive and Gram negative bacteria. Promiscuous is another term
used due to their ability to transfer by conjugation.

Molecular properties of plasmids:

 Compact conformation: It is supercoiled - can be nicked during DNA isolation so it

becomes less compact and if circular then it is open.
 Small high copy: random plasmid portioning – can be several plasmids in one cell,
can be small low copy.
 Large low-copy: 1-5 copies per cell directed plasmid partitioning. Its replication is
linked to chromosome replication. However, it does not require proteins from
chromosome.

Plasmids encode:
 Antibiotic resistance
 Metal/metalloid resistance – e.g. resistance to Hg and As.
 Virulence determinats – animal and plant pathogenicity.
 Bacteriocin production – molecules that kill other bacteria/microbes.
 Biodegradative capacities
 Symbiotic determinats- ability to fix nitrogen.

Transposon: DNA sequence with ability to move = “jumping genes”. It can be small of large. It
encodes genes for its own movement(?). Many eukaryotes have transposon.
Fitness cost of plasmid:

Plasmids confer energetic costs to hosts. If a plasmid provide fitness benefits it is stably maintained
by host. Plasmid and strain can co-evolve to decrease overall fitness cost to the strain

What do you need to be a good vector?

o Origin of replication (oriV)- must replicate in your host – understanding host range
important.
o Selection e.g. antibiotic resitance, Blue white colour selection.
o Multiple cloning sites(MCS)/ polylinker
o Promoter – can be inducible or constitutive.

Suicide Vector: a plasmid which is used with and oriV that is unable to replicate in host of interest.

Shuttle vector: a plasmid that contains two origins of replication allowing for replication in two
hosts.

Important!!: the size of plasmid will determine how to transfer into your host – transformation vs
conjugation. Check!
Control of gene expression
Operon: is a cluster of genes that code for proteins in the same metabolic pathway.

Lac operon (Lactose operon)

Lactose is cleaved by beta galactosidase into galactose and glucose.

Lac Operon is made up of different genes. It has a promoter, Lac I which synthesizes a repressor
which will be responsible of the expression of other genes in the operon.

lac Z: produces beta galactosidase

lac Y: produces lactose permease: it is a transporter that brings lactose in and out the cell.

lac A: thiogalactoside transacetylase: helps remove toxic metabolites. They are transported out by
lac Y protein.

Lac operon is an inducible operon which means that it is turn on when it is needed and off when it is
not.

In the absence of lactose, the repressor produced by lac I binds to the promoter and operator so the
proteins produced by the other genes are not generated due to the incapability of RNA polymerase
to bind the promoter. The active form of the repressor is a tetramer that binds the primary/
principal operator and the auxiliary operon so the RNA pol cannot bind DNA. This form of regulation
is known as negative regulation.

In the presence of lactose, the repressor is inactivated by lactose it has 2 binding sites: one for DNA
and one for lactose, however it can’t bind both at the same time. So once the repressor is inactive
the RNA polymerase can transcribe genes in operon. Notice that the repressor will be constitutively
expressed which means that it will be produced all the time as it has its own promoter and it is
essential for regulation.
There is a positive regulation of lac operon. It requires glucose as inhibitor. Glucose metabolism
does not require new proteins furthermore; this way of regulation is more efficient and it is called
catabolite repression.