BOCM 3714

Lecture 2

T: +27(0)51 401 9111 | [email protected] | www.ufs.ac.za

 Gene expression and replication:
an overview

• In 1958, Crick outlined

the synthesis of DNA,
RNA & proteins

• Central dogma of
molecular biology
– DNA directs own
replication
– DNA directs transcription
to yield RNA
– RNA directs translation to
yield proteins
 Protein synthesis: Translation

• Polypeptides are synthesised under direction of mRNA by

ribosomes

• Ribosomes:
– ⅔ RNA + ⅓ protein
– Size in prokaryotes = ~2500 kDa
– Size in eukaryotes = ~4200 kDa

• Ribosomal (rRNA) transcribed from DNA templates like all

kinds of RNA
Transfer RNA
• mRNA: series of nucleotide
sequences – codons
• Codons – specify amino
acid
• Codons DO NOT bind amino
acids
• Bind to transfer RNA (tRNA)
which are covalently linked
to amino acid
• tRNA - ~76 nt, trinucleotide
sequence – anticodon
• Anticodon – complementary
to codon
• Amino acid is covalently
linked to 3' end of tRNA –
aminocyl-tRNA: charging
• During translation – mRNA pass through ribosome
• Each codon bind to corresponding aminocyl-tRNA
• Ribosome transfers amino acid on tRNA to C-terminal end
of polypeptide chain
• Thus, polypeptide chain grows from N-terminus to C-
terminus
 The Genetic Code
• Nearly universal among all forms
of life – evidence of common
ancestor
• Express human genes in E. coli
• There are 4 bases, that can
occupy 3 positions in codon
• 43 = 64 possible codons
• 61 codons specify amino acids,
the other three STOP codons
(UAA; UAG; UGA)
• 2 amino acids (Met & Trp) only
one codon
• 3 amino acids (Leu, Ser, Arg) = 6
codons
• Genetic code = degenerate
• Most codons that specify a given
amino acid – synonyms: differ
only in 3rd nucleotide
 Aminocyl-tRNA synthetases

• Ribosome does not recognize the amino acid linked to

tRNA – but the anticodon
• Charging of tRNA with correct amino acid – critical for
accurate translation
• Enzymes that catalyses addition of amino acid to tRNA:
aminocyl-tRNA synthetases (aaRs)
• Cell contains 20 aaRs – one for each amino acid
• aaRs will charge all tRNAs that contain the codons for
corresponding amino acid
• aaRs must differentiate between tRNAs
• Many aaRs recognise anticodons, some recognize other
sites on tRNAs
 AUG codons

• Ribosomes read mRNA in 5' to 3' direction

• Initiation codon = AUG, Met

• tRNA that recognizes initiation codon, differs from tRNA

that delivers internal Met residues

• Both tRNAs charged by same methionyl-tRNA synthetase

Reading frame
• Correct reading frame
• Shift in one base –
result in completely
different protein
• How does ribosome
select initiation codon
among AUGs?
oProkaryotes: each
mRNA contains
sequence upstream (5'
end) that ribosome
recognise
oEukaryotes: initiating
codon – first AUG to
follow mRNA’s cap
 Post-translational modifications

• Often required for proteins to be functional

• The leading Met often removed by specific protease
• Various forms of post-translational modifications
– Specific protease cleavages
– Acylation
– Hydroxylation
– Methylation
– Phosphorylation
– Glycosylation (only eukaryotic proteins)
• Glycoproteins most common type of eukaryotic protein
 Molecular Cloning
• Require sufficient quantities of proteins of interest to study
specific aspects
• Development of molecular cloning – overcome these
restrictions
• Genetic engineering or recombinant DNA technology
• Definition: Clone is a collection of identical organisms
derived from single ancestor
Insights from bacteriophage lambda (λ)
cohesive sites
• In 1962, Allan Campbell noted that the linear genome of
bacteriophage λ forms a circle upon entering the host
bacterial cell by joining complementary single-stranded DNA
cohesive (cos) sites.

• The idea of joining DNA segments by “cohesive sites”

became a guiding principle in the development of
recombinant DNA technology.
 Restriction system

• First restriction endonuclease characterized in E.

coli K-12 by Matt Meselson and Bob Yuan.

• Bacteriophages propagate efficiently in K12, but

very low rate in related strain (strain B)

• Viral progeny of strain B propagate efficiently in

new host, but poorly in original host

• New host modify bacteriophage

What is molecular basis of host-specific
modification?

• Werner Arber showed that it results from

restriction-modification system in bacterial host

• System consists of a restriction endonuclease and

DNA methyltransferase
 Restriction Endonucleases

• Recognize a specific base sequence of 4-8 bases of dsDNA

• Cleave both strands
• DNA methyltransferases methylate a specific base in the
same base sequence recognized by the matched restriction
enzyme
• A restriction enzyme does not cleave its corresponding
methylated DNA
• Restriction-modification system protects bacterium against
foreign DNA
 Types of restriction endonucleases
• Four types
• Types I and III – carry both endonuclease & DNA
methyltransferase activity on single molecule
• Type I – cleave DNA at random site at least 1000bp from
recognition sequence
• Type III – cleave DNA at about 24-26bp from recognition
sequence
• Type IV – cleave methylated DNA
• Type II – endonuclease activity separate from DNA
methylation
– Discovered by Hamilton Smith and Daniel Nathans
– Cleave DNA within or close to recognition sequence
– Indispensable biochemical tools for DNA manipulation
• Type II restriction enzyme recognition sites – exact twofold
symmetry: palindrome (noon, level, madam)
• Cleave DNA symmetrically – produce cohesive or sticky ends
• Cleave at twofold axis – blunt ends
Names of restriction enzymes

EcoRI

E = Escherichia (genus)
co = coli (species)
R = strain or serotype
Roman number – number of enzyme
identified from bacterium
 • Digestion of DNA
molecule with
restriction enzyme –
precisely defined
fragments
• Separated by gel
electrophoresis
according to size
• Generate restriction
map
RFLP is a difference in the size of DNA restriction
fragment (restriction map) between individuals. It can
serve as a useful genetic marker for the analysis and
mapping of a large genome. RFLP is based on the
principle that small differences in the DNA sequence
can alter restriction enzyme cutting patterns.
• Individuality in species – derived from genetic polymorphism
• Human chromosome differ in sequence every ~1250bp:
create and eliminate restriction enzyme sites
• Obtain different patterns: restriction-fragment length
polymorphisms (RFLPs)
 Cloning vectors

• Plasmids, viruses & artificial chromosomes

1. Plasmid-based cloning vectors

– Extrachromosomal double-stranded circular DNA
– Few copies per cell – stringent control (“low copy”)
– 10-700 copies per cell – relaxed control (“high copy”)
– 1. Carry origin of replication (ori), can replicate
autonomously in bacteria or yeast
– 2. Can provide resistance to antibiotics to host bacteria
– 3. Polylinker (multiple cloning site) – segment of DNA
containing various restriction enzyme recognition sites
 Transformation

• Host bacterium takes up plasmid when two are

mixed
– Process enhanced through the presence of divalent
cations such as Ca2+
– Cations interact with negatively charged phosphates in
phospholipid membrane, destabilize membrane,
creating transient pores
– Brief heating to ~42°C (heat shock)
– Foreign DNA taken up

• Bacterium: transformation competent

 2. Virus-based cloning vectors
• Bacteriophage λ
– Part of genome not required for infection: replace with
foreign DNA
– Can be used to clone up to 16 kb
• Cosmid vector: no phage genes, reproduce as plasmids
– Can accommodate inserts up to 49 kb
– Foreign DNA inserted between cos sites (complementary
single-stranded DNA cohesive)
• Filamentous bacteriophage M13
– Single-stranded circular DNA, small inserts (about 1kb)
• Baculoviruses
– Viruses infecting insects
– Can be replicated safely in insect cell cultures in lab
• Lentiviral or adenoviral vectors
– Mammalian cells
 3. YAC, BAC & MAC

• Yeast artificial chromosome, bacterial artificial

chromosome & mammalian artificial
chromosomes

• Contains:
– Autonomously replicating sequence (ARS)
– Centromere (attach to spindle during mitosis & meiosis)
– Telomeres (permits replication)
 Summary
• Translation – components
– Genetic code
– Aminoacyl-tRNA synthetases
– Reading frame

• Molecular Cloning
• Restriction enzymes
– Restriction modification
– Application in recombinant DNA technologies
• Cloning vectors
– Plasmids
• Transformation