Download biological background the central dogma of molecular biology

BIOLOGICAL BACKGROUND
¨ Central Dogma
¨ DNA and RNA Structure
¨ Replication, Transcription and Translation
¨ Techniques of Molecular Genetics
• Using restriction enzymes
• Using PCR
THE CENTRAL DOGMA OF MOLECULAR BIOLOGY
Genetic information flow:
1) From DNA to DNA during
its transmission from
generation to generation.
2) From DNA to Protein during
its phenotypic expression in
an organism.
Transcription: DNA to
RNA (sometimes
reversible).
Translation: RNA to
protein (irreversible).
Occassionally, genetic
information flows from RNA to
DNA (reverse transcription).
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HEREDITARY MATERIAL
Major structural differences between
DNA and RNA are:
1) Hydrogen vs. Hydroxide
2) Thymine (T)
vs. Uracil (U)
Most organisms and viruses have
DNA as their hereditary material.
Some viruses have RNA (single
and double stranded).
BUILDING BLOCKS
• DNA molecules are typically composed of two
strands that are related through
complementary base pairs (Hydrogen bonds).
• Phosphodiester links:
Nucleotides are linked
together by
phosphodiester
backbone.
Anti-parallel strands
Purines Æ A, G
Pyrimidine Æ C, T (U)
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TYPES OF RNA
Four different classes of RNA molecules play essential roles in gene
expression:
• Messenger RNA (mRNA): intermediaries that carry genetic
information from DNA to the ribosomes where proteins are synthesized.
• Transfer RNA (tRNA): small RNA molecules that function as adaptors
between amino acids and the codons in mRNA during translation.
• Ribosomal RNA (rRNA): structural components of the ribosomes, the
intricate machines that translate nucleotide sequences of mRNAs into
amino acid sequences of polypeptides.
• Small Nuclear RNA (snRNA): structural components of spliceosomes,
the nuclear structures that excise introns from nuclear genes.
ORGANISMS
Organisms (including bacteria and
blue-green algae) that lack true
nuclei in their cells and that do
not undergo meiosis
Coupled transcription and
translation
Organisms that have nuclei
enclosed by a membrane within
their cells
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CHROMOSOMES
Prokaryotic chromosome:
• Most contain a single, double stranded, circular DNA molecule.
• The DNA is mostly naked, but is supercoiled and looped.
Eukaryotic chromosome:
• Consist of very long, linear double stranded DNA.
• The DNA is complexed with twice as much protein (histones
organized in nucleosomes).
• The protein helps compact it into the nucleus.
GENE
¾ Region of DNA that controls a
hereditary characteristic.
¾ It usually corresponds to a sequence
used in the production of a specific
protein or RNA.
¾ A gene carries biological information in
a form that must be copied and
transmitted from each cell to all its
progeny. This includes the entire
functional unit: coding DNA sequences,
non-coding regulatory DNA sequences,
and introns.
¾ Genes can be as short as 1000 base
pairs or as long as several hundred
thousand base pairs.
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GENE
Intron and Exon
Exon: segment of a
eukaryotic gene that
corresponds to the
sequences in the final
processed RNA transcript of
that gene. In some species
(including humans) exons are
separated by long regions of
DNA (introns).
Intron: Intervening
sequences of DNA bases
within eukaryotic genes that
are not represented in the
mature RNA transcript
because they are spliced out
of the primary RNA
transcript.
TRANSFER OF GENETIC INFORMATION
Perpetuation of genetic information from generation to generation
DNA
Control of
the
phenotype:
Gene
expression
T T T
.. .. ..
A A A
Transcription
DNA-dependent
RNA polymerase
Replication
DNA-dependent
DNA polymerase
DNA
DNA
T T T
.. .. ..
A A A
T T T
.. .. ..
A A A
Reverse transcription
RNA-dependent DNA polymerase
(reverse transcriptase)
U U U
mRNA
Translation
Complex process
involving ribosomes,
tRNA and other
molecules
Polypeptide
(not folded)
phe
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REPLICATION
Essential components: 1) template 2) dNTP 3) DNA polymerase 4) primer
¾ To reproduce, a cell must copy
and transmit its genetic
information (DNA) to all of its
progeny.
¾ DNA replicates, following the
process of semiconservative
replication.
¾ Each strand of the original
molecule acts as a template for the
synthesis of a new complementary
DNA molecule following the rules
of complementary base pairing:
adenine (A) to thymine (T) and
guanine (G) to cytosine (C).
REPLICATION
When DNA replicate, many different proteins work together to accomplish the
following steps:
1) The two parent strands are unwound with the help of DNA helicases.
2) Single stranded DNA binding proteins attach to the unwound strands,
preventing them from winding back together.
3) The strands are held in position, binding easily to DNA polymerase, which
catalyzes the elongation of the leading and lagging strands (DNA polymerase also
checks the accuracy of its own work).
4) DNA polymerase requires a primer (DNA or RNA).
5) DNA polymerase on the leading strand can operate in a continuous fashion.
6) RNA primer is needed repeatedly on the lagging strand to facilitate synthesis of
Okazaki fragments (DNA primase helps to build the primer).
7) Each new Okazaki fragment is attached to the completed portion of the lagging
strand in a reaction catalyzed by DNA ligase.
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REPLICATION
- Replicate DNA in the 5’ Æ 3’ direction
- Also has 3’ Æ 5’ exonuclease activity that
eliminates RNA primers on lagging strand
Continuous
synthesis
Unwinds DNA
Discontinuous
synthesis
Lays down RNA primer
TRANSCRIPTION
RNA-polymerase-catalyzed synthesis of RNA from a DNA template.
Transcription Proceeds in three distinct phases:
1. Initiation (binding of RNA polymerase to template DNA).
2. Elongation (nucleotides are added to the DNA template).
3. Termination (the enzyme and RNA is release from DNA template).
Initiation: occurs in a specific region on the DNA called the promoter.
• The promoter contains sequence elements that bind several transcription factors in
combination with RNA polymerase and indicate the first base to be copied into an RNA
transcript (the promoter also includes sequences involved in regulation of transcription).
• Several transcription factors called initiation factors are necessary for RNA polymerases to
recognize and bind tightly to the promoter (no primer is necessary).
• The initiation factors bind first to the promoter, forming an active complex that fits one or
more sites on the RNA polymerase enzyme. The enzyme then binds tightly to the initiation
factors and the promoter, and the DNA unwinds in their promoter region.
• Of the two DNA nucleotide chains fully exposed by the unwinding, only one contains the
correct promoter sequences and acts as a template. The opposite, non-template chain is
complementary to the promoter and template but does not contain encoded information
(different genes may have their template chains on either side of the DNA helix).
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TRANSCRIPTION (continued)
Elongation:
• Once the first base is added, elongation begins and RNA nucleotides add sequentially until
the polymerase reaches the end of the template (30 – 50 nucleotides per second).
Termination:
• Sequences signaling termination
also appear in some eukaryotic
genes, such as those encoding
rRNAs.
• In eukaryotic mRNA genes,
termination may be coupled to
processing reactions rather than
occurring in response to specific
signal sequences in the DNA.
• Termination factors contribute
to separation of RNA polymerase
and elongating factors from the
template and release of the
transcript in prokaryotes and
possibly also in eukaryotes.
http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm
RNA PROCESSING: PRE-mRNA Æ mRNA
• Primary transcripts produced in the nucleus must undergo processing steps to produce
functional RNA molecules for export to the cytosol:
1) Synthesis of the cap: modified G
which is attached to the 5’ end of the
pre-mRNA (cap protects the RNA from
being degraded by enzymes).
2) Removal of introns: splicing of
nuclear transcripts involves particular
sequence signals near splice junctions
and is conducted by snRNA.
3) Synthesis of the poly(A) tail:
transcript is cut at a site when
completed, and a poly(A) tail is
attached to the exposed 3’ end.
4) mRNA is now exported to the
cytosol.
¾ In most mammalian cells, only 1% of the DNA
sequence is copied into a functional RNA (mRNA).
5) The remainder of the original
transcript is degraded and the RNA
polymerase leaves the DNA.
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REVERSE TRANSCRIPTION
¾ The process of making a segment of DNA from a segment of RNA utilizing a
reverse transcriptase enzyme that reads from 3’ to 5’ (naturally occurs in
retroviruses).
¾ The newly formed single stranded DNA is complementary, by virtue of the rules
of base pairing, to the originally isolated mRNA (Complementary DNA - cDNA).
¾ cDNA represents only the exons or coding region of the gene.
¾ A poly dT linker-primer is often used for reverse transcription.
mRNA G U A A U C C U C
Reverse
transcriptase
cDNA
TT
AG
GA
G
CA
TTA G
G
AG
C
GA
C A TATTATGG G
GA
C A T TAA A
GG
G GA G G
T
AA
CC
AT A T
A
GG
GG
A
GG
T
T
A
AA
TT
GG
GG
AGA
GG
TA
TA
C AC C
TRANSLATION
¾ The ribosome binds to the mRNA at the start
codon (AUG) that is recognized only by the
initiator tRNA.
¾ The ribosome proceeds to the elongation
phase of protein synthesis.
¾ Complexes, composed of an amino acid
linked to tRNA, sequentially bind to the
appropriate codon in mRNA by forming
complementary base pairs with the tRNA
anticodon.
¾ The ribosome moves from codon to codon
along the mRNA.
¾ Amino acids are added one by one,
translated into polypeptidic sequences dictated
by DNA and represented by mRNA.
¾ At the end, a release factor binds to the stop
codon, terminating translation and releasing
the complete polypeptide from the ribosome.
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THE GENETIC CODE
One specific amino acid can correspond to more than one codon.
Key
Amino Acid
Key
Amino Acid
Ala
Alanine
Asp
Aspartic acid
Phe
Phenylalanine
His
Histidine
Lys
Lysine
Met
Methionine
Pro
Proline
Arg
Arginine
Thr
Threonine
Trp
Tryptophane
Cys
Cysteine
Glu
Glutamic acid
Gly
Glycine
Ile
Isoleucine
Leu
Leucine
Asn
Asparagine
Gln
Glutamine
Ser
Serine
Val
Valine
Tyr
Tyrosisne
CONTROL OF GENE EXPRESSION
Examples of regulation at each of the steps are known, although for most genes
the main site of control is step 1: transcription of a DNA sequence into RNA
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TECHNIQUES OF MOLECULAR GENETICS
The action of restriction enzymes:
• Restriction enzymes (EcoRI in this
example) , surrounds the DNA
molecule at the point it
seeks(sequence GAATTC).
• It cuts one strand of the DNA double
helix at one point and the second
strand at a different, complementary
point (between the G and the A base).
• The separated pieces have single
stranded "sticky-ends," which allow
the complementary pieces to combine.
• The newly joined pieces are
stabilized by DNA ligase.
SOUTHERN BLOTTING
Detection of specific DNA fragments by gel-transfer hybridization
1)
2)
3)
4)
5)
6)
7)
8)
9)
The mixture of double-stranded DNA fragments generated by restriction nuclease
treatment of DNA is separated according to length by electrophoresis.
A sheet of either nitrocellulose paper or nylon paper is laid over the gel, and the separated
DNA fragments are transferred to the sheet by blotting.
The gel is supported on a layer of sponge in a bath of alkali solution, and the buffer is
sucked through the gel and the nitrocellulose paper by paper towels stacked on top of the
nitrocellulose.
As the buffer is sucked through, it denatures the DNA and transfers the single-stranded
fragments from the gel to the surface of the nitrocellulose sheet, where they adhere firmly.
(This transfer is necessary to keep the DNA firmly in place while the hybridization
procedure is carried out).
The nitrocellulose sheet is carefully peeled off the gel.
The sheet containing the bound single-stranded DNA fragments is placed in a sealed
container together with buffer containing a radioactively labeled DNA probe specific for the
required DNA sequence.
The sheet is exposed for a prolonged period to the probe under conditions favoring
hybridization.
The sheet is removed from the container and washed thoroughly, so that only probe
molecules that have hybridized to the DNA on the paper remain attached.
After autoradiography, the DNA that has hybridized to the labeled probe will show up as
bands on the autoradiograph.
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SOUTHERN BLOTTING
• An adaptation of this technique to detect
specific sequences in RNA is called Northern
blotting. In this case mRNA molecules are
electrophoresed through the gel and the probe is
usually a single-stranded DNA molecule.
• Northern blots allow investigators to determine
the molecular weight of an mRNA and to measure
relative amounts of the mRNA present in different
samples.
INSERTING DNA INTO A PLASMID
• Plasmid vectors are small circular molecules of
double stranded DNA derived from natural
plasmids that occur in bacterial cells.
• A piece of DNA can be inserted into a plasmid if
both the circular plasmid and the source of DNA
have recognition sites for the same restriction
endonuclease.
• The plasmid and the foreign DNA are cut by this
restriction endonuclease producing intermediates
with sticky and complementary ends.
• Those two intermediates recombine by basepairing and are linked by the action of DNA ligase.
• Few mismatches occur, producing an undesirable
recombinant.
• The new plasmid can be introduced into bacterial
cells that can produce many copies of the inserted
DNA .
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CLONING INTO A PLASMID
• Plasmid is used to import recombinant DNA into a host cell
for cloning.
• DNA fragment that contains a gene of interest is inserted
into a cloning vector or plasmid.
• The plasmid carrying genes for antibiotic resistance, and a
DNA strand, which contains the gene of interest, are both cut
with the same restriction endonuclease.
• The plasmid is opened up and the gene is freed from its
parent DNA strand. They have complementary "sticky ends."
• The opened plasmid and the freed gene are mixed with DNA
ligase, which reforms the two pieces as recombinant DNA.
• Plasmids + copies of the DNA fragment produce quantities of recombinant DNA.
• This recombinant DNA stew is allowed to transform a bacterial culture, which is then exposed to
antibiotics.
• All the cells except those which have been encoded by the plasmid DNA recombinant are killed,
leaving a cell culture containing the desired recombinant DNA.
• DNA cloning allows a copy of any specific part of a DNA (or RNA). This technique is the first stage of
most of the genetic engineering experiments: production of DNA libraries, PCR, DNA sequencing, etc.
CONSTRUCTION OF GENOMIC or cDNA LIBRARY
¾ A genomic library comprises a set of
bacteria, each carrying a different small
fragment of genomic DNA or cDNA.
¾ For simplicity, cloning of just a few
representative fragments (colored) is
shown.
¾ In reality, all the gray DNA fragments will
also be cloned.
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SCREENING A GENOMIC or cDNA LIBRARY
DNA probes or antibodies are used to screen
libraries for specific DNA sequences:
• Transform E. Coli with a genomic library.
• Plate on selective growth medium; colonies grow.
• Replica plate colonies transfer onto new selective
medium plate with membrane filter or surface;
colonies grow on filter.
• Filter removed from culture dish, bacteria lysed, DNA
denatured and bound to filter.
• Probe DNA hybridized to DNA on filter.
• Wash filter free of unbound probe. Detect
hybridization by autoradiography for radioactively
labeled probes.
PCR
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QUANTITATIVE RT-PCR
• Real-time reverse-transcriptase (RT) PCR quantifies the initial amount
of the template.
• Real-time PCR monitors the fluorescence emitted during the reaction
as an indicator of amplicon production during each PCR cycle (i.e., in
real time) as opposed to the endpoint detection by conventional
quantitative PCR methods.
• The real-time PCR system is based on the detection and
quantification of a fluorescent reporter. This signal increases in direct
proportion to the amount of PCR product in a reaction.
• By recording the amount of fluorescence emission at each cycle, it is
possible to monitor the PCR reaction during exponential phase where
the first significant increase in the amount of PCR product correlates to
the initial amount of target template.
REFERENCES
Principles of Genetics. Ed.: D. P. Snustad, M. J. Simmons and J. B. Jenkins.
John Wiley & Sons, Inc., New York
http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm
http://www.accessexcellence.org/AB/GG/
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