Some basic molecular biology
Summaries of: Replication, Transcription;
Translation, Hybridization, PCR
Material adapted from
Lodish et al, Molecular Cell Biology, 4th ed,
Freeman, 2000; some figures from Genes VII by
Lewin, Oxford, 2000; Alberts et al, Essential Cell
Biology, An Introduction to the Molecular Biology of
the Cell, Garland, 2000; and elsewhere.
DNA Replication
The regular pairing of bases in the double helical DNA structure
suggested to Watson and Crick a mechanism of DNA synthesis,
Their proposal that new strands of DNA are synthesized by
copying of parental strands proved to be correct.
The DNA strand that is copied to form a new strand is called a
template. The information in the template is preserved: although
the first copy has a complementary sequence, not an identical
one, a copy of the copy produces the original (template) sequence
again. In the replication of double-stranded or duplex DNA
molecule, both original (parental) DNA strands are copied. When
copying is finished, the two new duplexes, each consisting of one
of the original strands plus its copy, separate from each other.
DNA Replication, ctd
All DNA synthesis proceeds in the same chemical direction: 5’->3’.
Nucleic acid chains are assembled from 5’ triphosphates of
deoxyribonucleosides. Strand growth is energetically unfavourable,
but is driven by the energy available in the triphosphates.
The enzymes that copy (replicate) DNA to make more DNA are
DNA polymerases.
DNA polymerases cannot initiate chain synthesis de novo; instead
they require a short preexisting DNA strand, called a primer, to
begin chain growth. With a primer base-paired to the template
strand, a DNA polymerase adds nucleotides to the free hydroxyl
group at the 3’ end of the primer.
Replication of duplex DNA requires assembly of many proteins (at
least 30) at a growing fork: helicases to unwind, primases to prime,
ligases to ligate (join), topisomerases to remove supercoils, RNA
polymerase, etc.
Transcription (mainly in prokaryotes)
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Transcription is a very complex process involving many steps and
many proteins.
RNA polymerase initiates transcription of most genes at a unique
position (a single base: the start site) in the template DNA upstream
of the coding sequence.
RNA polymerase binds to specific promoter sequences to initiate
transcription
Initiation begins when a subunit of a polymerase binds to a promotor
DNA sequence
Repressors bind to DNA sequences called operators which overlap the
promoter region that contacts RNA polymerase. A bound repressor
interferes with binding of RNA polymerase and transcription initiation
Activators generally bind to DNA on the opposite side of the helix
from the polymerase at specific positions
Transcription, ctd
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The activator cAMP-CAP stimulates transcription by forming a
complex with RNA polymerase that has a greater affinity for DNA
sites than the individual proteins
In eukaryotes, the situation is more complex still.
Expression of eukaryotic protein coding genes is regulated through
multiple transcriptional control regions
Regulatory elements are often many kilobases from start sites
There are three different types of RNA polymerases with different
functions
Enhancers, usually 100-200 bp in length contain multiple 8-20 bp
control elements, may be from 200 bp to tens of kilobases upstream
of downstream from a promoter, within an intron, or downstream from
a final exon of gene
Promoter-proximal elements and enhancers often are cell-type specific
From Alberts
et al, 1998
From Genes VII
Translation
• The AUG start codon is recognized by methionyl-tRNAiMet
• Eukaryotic initiation of protein synthesis occurs at th 5’
end and internal sites in mRNA
• During chain elongation each incoming aminoacyl-tRN A
moves through three ribosomal sites
• This process is catalyzed by proteins known as elongation
factors
• Protein synthesis is terminated by a release factor when a
stop codon is reached
• In eukaryotic cells, multiple ribosomes are commonly
bound to a single mRNA, forming a circular polysome.
• As each ribosome completes translation and is released
from the 3’ end of the mRNA, the subunits probably
reassemble quickly at the 5’ end.
Hybridization
This is a technique which exploits a potent
feature of the DNA duplex - the sequence
complementarity of the two strands.
Remarkably, DNA can reassemble with
perfect fidelity from the separated strands.
The melting temperature of a DNA
molecule is denoted Tm and this depends on
G+C content and salt concentration.
The Southern blotting technique
Polymerase Chain Reaction
This reaction is used to amplify specific DNA sequences in a
complex mixture when the ends of the sequence are known. The
source is heat-denatured into single strands. Two synthetic
oligonucleotides complementary to the 3’ ends of the segment of
interest are added in great excess to the denatured DNA and the
temperature is lowered to 50-60˚C or even lower. The genomic
DNA remains denatured, because the complementary strands are
at too low a concentration to encounter each other during the
period of incubation, but the specific oligonucleotides, which are
at a very high concentration, hybridize with their complementary
sequences in the genomic DNA.
PCR, ctd
The hybridized oligos then serve as primers for DNA chain
synthesis, which begins upon addition of a supply of
dNTPs and a temperature resistant polymerase such as that
from Thermus aquilus (a bacterium that lives in hot
springs). This enzyme, called Taq polymerase, can extend
primers at temperatures up to 72˚C. When synthesis is
complete, the whole mixture is heated further (to 95˚C) to
melt the newly formed duplexes. When the temperature is
lowered again, a new round of synthesis takes place
because excess primer is still present. Repeated cycles of
synthesis (cooling) and melting (heating) quickly amplify.