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• A Molecular Biology primer…..
• Genetic information is carried on nucleic
acids - DNA &RNA
• Genetic information is carried on nucleic
acids - DNA &RNA
The five bases are:
adenine, guanine = purines
• A Molecular Biology primer…..
• Genetic information is carried on nucleic
acids - DNA &RNA
The five bases are:
adenine, guanine = purines
and...
thymine, cytosine, uracil = pyrimidines
A = T (or A = U)
C
G
• Bases: adenine, guanine, cytosine, uracil,
thymine
• Nucleosides: = base + sugar
– on RNA sugar is ribose
– adenosine, guanosine, cytidine, uridine, thymidine
• on DNA sugar is deoxyribose
• deoxyadenosine, deoxyguanosine etc...
• Nucleotides: base + sugar + phosphate
• RNA:
• adenylate, guanylate, cytidylate, uridylate
• Nucleosides: base + sugar + phosphate
• RNA:
• adenylate, guanylate, cytidylate, uridylate
• DNA:
• deoxyadenylate, deoxyguanylate etc...
• By convention, nucleic acid sequence refers
to bases not nucleotides.
• By convention, nuceic acid sequence refers
to bases not nucleotides.
• Sequence is always written 5’
3
• Native DNA is normally double stranded
• Native DNA is normally double stranded
• …the bases project inwards and are
attracted by H-bonding.
• Native DNA is normally double stranded
• …the bases project inwards and are
attracted by H-bonding.
• RNA is normally single stranded.
• At a particular temperature (Tm) H-bonds
are broken and structure comes apart.
• At a particular temperature (Tm) H-bonds
are broken and structure comes apart.
… = “melting” (or denaturation)
• Cooling will lead to renaturation of
structure.
• Cooling will lead to renaturation of
structure
… = annealing.
• Cooling will lead to renaturation of
structure
… = annealing.
= basis in vitro of:
hybridisation;
PCR
Information flow is in one direction
only:
transcription
DNA
translation
RNA
Protein
Information flow is in one direction
only:
transcription
DNA
translation
RNA
retroviruses
Protein
Information flow is in one direction
only:
transcription
DNA
translation
RNA
retroviruses
Enzyme activity
Protein
Modifications:
Cleavage:
+
Modifications:
Cleavage:
Ligation:
+
+
Modifications:
Cleavage:
+
(exo / endonucleases)
Ligation:
+
(ligases)
Splicing:
+
• DNA – two major functions:• Replication and transcription
• DNA – two major functions:• Replication and transcription
• …in each case a template strand is needed.
• In replication, the duplex splits locally and
each strand acts as a template for synthesis
of a new strand.
• In transcription, the anti-sense strand
acts as a template.
• In transcription, the anti-sense strand
acts as a template.
• Therefore the RNA sequence is identical
to the coding strand
•
RNA produced is the primary transcript.
1. 5’ capping with 7-methyl guanosine
•
RNA produced is the primary transcript.
1. 5’ capping with 7-methyl guanosine
2. 3’ cleavage to “poly A site”
•
RNA produced is the primary transcript.
1. 5’ capping with 7-methyl guanosine
2. 3’ cleavage to “poly A site”
3. polyadenylation (~250 A)
•
RNA produced is the primary transcript.
1.
2.
3.
4.
5’ capping with 7-methyl guanosine
3’ cleavage to “poly A site”
polyadenylation (~250 A)
Splicing of exons by excision of introns.
•
RNA produced is the primary transcript.
1.
2.
3.
4.
5.
5’ capping with 7-methyl guanosine
3’ cleavage to “poly A site”
polyadenylation (~250 A)
Splicing of exons by excision of introns.
… = mRNA
•
RNA produced is the primary transcript.
1.
2.
3.
4.
5.
5’ capping with 7-methyl= pre-mRNA
guanosine
3’ cleavage to “poly A site”
Or heterogeneous
nuclear RNAs (hnRNA)
polyadenylation (~250 A)
Splicing of exons by excision of introns.
… = mRNA
• Note that mRNA and pre-mRNA contain
untranslated regions
– 3’ UTRs
– 5’ UTRs
• Note that mRNA and pre-mRNA contain
untranslated regions
– 3’ UTRs
– 5’ UTRs
…these are “outside” the exon sequences.
• Splicing is a variable process
• Dogma: 1 gene
1 polypeptide
• Splicing is a variable process
• Dogma: 1 gene
1 polypeptide
• In fact: many products can be produced
from each gene.
• Splice variants: removal of selected exons.
• Splice variants: removal of selected exons.
• Another reason….
• Another reason….
• Post-translational modification:
• Post-translational modification:
–
–
–
–
Proteins
Glycoproteins
Phosphoproteins
lipoproteins
• Post-translational modification:
–
–
–
–
Proteins
Glycoproteins
Phosphoproteins
Lipoproteins
– The proteome is much more descriptive than
the genome.
• Translation
• There are 20 amino acids used in protein
synthesis, but only 4 different bases.
• Translation
• There are 20 amino acids used in protein
synthesis, but only 4 different bases.
…. So how does base sequence code for the
amino acid sequence?
• Each amino acid is coded for by 3 bases
= a codon (a triplet of bases)
• Each amino acid is coded for by 3 bases
= a codon (a triplet of bases)
Translation is co-linear
• Each amino acid is coded for by 3 bases
= a codon (a triplet of bases)
Translation is co-linear
… i.e. codon sequence matches the final aa
sequence.
– CACAACCAAUUU
– his asn gln phe
– CACAACCAAUUU
– his asn gln phe
– “Start” and “stop” codons define the reading
frame (coding region)
– CACAACCAAUUU
– his asn gln phe
– “Start” and “stop” codons define the reading
frame (coding region)
…. Which can be shifted
- physiologically
- by deletion / insertion mutations
ORF = open reading frame
ORF = open reading frame
= a suspected coding region once start / stop
codons identified.