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Central Dogma & PCR
B91901070
Wang Yu-Hsin
Central Dogma
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What is Central Dogma?
DNA
RNA
Protein
transcription
translation
All cells express their genetic information in this way.
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Cells must control the amount of protein
according to their demand.
How do they regulate the efficiency ?
Transcription
(from DNA to RNA)
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They use the same language, nucleotide, to
store information.
Differences between DNA and RNA
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Deoxyribonucleotide v.s ribonucleotide
Base pairs:
A = T & C≡G in DNA; T is replaced by U in RNA.
Structure in a cell:
DNA: Double-stranded helix
RNA: Single-stranded with folded shape.
Transcription
(from DNA to RNA. Cont’d)
1. Open and unwind a small portion of
the DNA double-stranded helix. Choose
one strand as the template.
2. Produce RNA, with elongating one
nucleotide at a time from 5’end to 3’ end
and have the sequence exactly
complementary to the template.
Transcription
(from DNA to RNA. Cont’d)
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There are some differences between RNA transcription and
DNA replication.
Size of nucleotide sequence.
RNA strand does not remain H bond to the DNA template and
quickly releases from the DNA as it synthesize for efficient
RNA production.
RNA polymerase can catalyze the formation of
phosphodiester bond( RNA’s sugar-phosphate backbone).
But it dose not check the correctness of previous base-pair,
which means that RNA transcript can tolerate high error rate.
How do they find the site to start or to finish?
Transcription
(RNA Polymerase)
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RNA polymerase has to recognize the start of the
gene. The initiation of transcription is very important,
for it regulates which protein to be produced.
Sigma factor in RNA polymerase takes the
responsibility of recognizing promoter DNA.
After RNA polymerase latches on the DNA and
synthesizes about ten nucleotides, sigma factor
releases from it.
After transcription stops, RNA polymerase leaves
and reassociate with a free sigma factor.
Then polymerase finds a new promoter…
Transcription
(in a procaryotic cell)
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- RNA polymerase first weakly sticks to DNA when it
meet the DNA.
- Then, polymerase slides along the DNA…
- Polymerase finds promoter and latch on the DNA
tightly.
- Open DNA double-stranded helix and choose one
strand as the template. Transcribe RNA by base
paring.
- When the polymerase encounters the terminal,
transcription stop.
- Release both the DNA template and newly made
RNA.
How do eucaryotes work?
Transcription
(Procaryotes v.s Eucaryotes)
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Bacteria do not have nucleus, so fresh RNA
which are just transcribed can contact
rebosomes in the cytoplasm directly.
Bacterial mRNA can contain the instructions
for several different proteins.
Genes are close to each other.
Bacterial mRNA degrade rapidly.( about 3
min)
Transcription
(Procaryotes v.s Eucaryotes. Cont’d)
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For eucaryotes, newly transcribed
RNA( primary RNA), inside the nucleus,
needs to take some processes of
modification before entering the cytoplasm.
Capping at 5’ end with a methyl G.
Splicing and remove introns( non-coding
sequences).
Polyadenylation at 3’end.( poly-A tail)
Transcription
(Procaryotes v.s Eucaryotes. Cont’d)
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In introns there are some short nucleotide
sequences as cues for its removal.
Splicing enzyme: snRNPs, which usually
assemble when catalysis begins.
With different splicing method, different
mRNA forms. This gene recombination
provides evolutionary flexibility. Also, the life
time of eucaryotic mRNA varies.
Translation
(from RNA to Protein)
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They use different languages. mRNA is made
up of nucleotides, whereas protein is
sequences of amino acid.
There are four types of nucleotides; however,
there are 20 types of a.a.
Three consecutive nucleotides in mRNA as
codon. => 64 combinations
Genetic codes are general rules for the
present-day organism.
Translation
(tRNA with amino acid)
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tRNA, transfer RNA
About 80 nucleotides long, with 3D structure.
Amino acid attaches to 3’ end.
Translation
(tRNA with amino acid. Cont’d)
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Redundancy. There are 61 types of codon,
31 types of tRNA, 20 types of amino acid.
Different tRNAs can carry the same type of a.a.
2. tRNA can do base-pairing with different mRNA
codons. This depends on constructure and
means that tRNA can tolerate mismatch
(wobble).
- Wobbles are alternative codons for a.a, different
only in their third nucleotide.
1.
Translation
(tRNA with amino acid. Cont’d)
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Aminoacyl-tRNA synthetases: for recognition
and attachment of the correct a.a, covalently
couple each a.a to its appropriate set on
tRNA with high energy bond.
Each kind of amino acid has the specific
synthetases of its own.
To synthesize protein, a cell still needs some
place for manufacture...
Translation
(Ribosomes for protein making)
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Ribosome: The protein manufacturing machine.
Made up of proteins and rRNA.
Combination of the large subunit and the small
subunit, both made in the nucleus and then sent
back to the cytoplasm.
Most found in cytosol and ER.
Three binding site for tRNA: E, P, and A site. During
the process of protein synthesis, no more than two
sites contain tRNA mocules at a time.
If needed, two subunits come together on mRNA. After
protein synthesis complete, they separate.
A tRNA carrying the next aa. In the
chain bound to A site.
Break high energy bond between tRNA
and a.a. and form peptide bond in the
large subunit.
Then... Back to step 1.
＊What if the initiation site is wrong…
Translation
(Condons for initiation)
Translation
(Condons for terminal & others)
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UAA, UAG, and UGA don’t specify any
amino acid.
Release factor protein bind to A-site, no more
peptidyl transfer but addition of H2O.
Free carboxyl end of the growing polypeptide
chain.
Folding.
Polyribosmes.
PCR
(Polymerase Chain Reaction)
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Amplify selected DNA or mRNA sequence of small
amount.
Entirely in vitro.
Based on the use of DNA polymerase to copy a
DNA template in repeated rounds of replication.
High sensitivity.
Polymerase is guide to the sequence to be copied by short primer
oligonucleotides.
PCR can be used only to clone DNA whose beginning and end are
known.
(1) 90ºC, open the DNA doublestranded helix.
(2) 50ºC, hybridization with primer.
(3)75ºC, DNA synthesize.
Repeating for 20~30 times.
The amount doubles in every cycle.
Only the sequence bracketed by two
primers is amplified!
Reference.
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Essential Cell Biology, by Alberts et al., 1998,
Garland
Some pictures are copied from wikipedia and
http://www.geneticengineering.org
THE END
Thanks for your attention