1. Central Dogma & PCR B
Wang Yu-Hsin

2. Central Dogma What is Central Dogma? DNA RNA Protein
transcription translation
　All cells express their genetic information in this way.
Cells must control the amount of protein according to their demand.
How do they regulate the efficiency ?
不讓DNA直接當作模板以合成蛋白質 控量

3. Transcription (from DNA to RNA)
They use the same language, nucleotide, to store information.
Differences between DNA and RNA
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.
RNA’s 3D structure has structural and catalytic function, whereas DNA just stores genetic info.

4. 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.
Choose one strand as template: polymerase必須依照promoter( asymetric)的方向作合成
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.

5. Transcription (from DNA to RNA. Cont’d)
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?
RNA is much shorter than DNA!(RNA 約只有幾千個核甘酸,而DNA超過250 million nucloetide pairs)
RNA轉錄約1,500nucleotide in 50 sec.
Error rate:0.01% for RNA; DNA: 10^-7, DNA存有genetic info, cannot stand errors.
功能不同,RNA不需要這麼accurate. 3.

6. Transcription (RNA Polymerase)
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…

8. Transcription (in a procaryotic cell)
- 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?

9. Transcription (Procaryotes v.s Eucaryotes)
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)
1. Bacteria, mrna translation usually take place before mrna transcription complete.
2.Most proteins are encoded by an uninterrupted stretch of DNA sequence. So newly transcribed rna can serve as mrna without further processing.
3.Degradation of mrna affect the amount of the protein. After that, mrna are separated into nucleotides.
** Without intron, bacteria can reproduce quickly for shorter genome.
(需要快速繁殖的真核細胞的intron也較少較短)

10. Transcription (Procaryotes v.s Eucaryotes. Cont’d)
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)
Capping通常在轉錄完整條rna前就進行了. G的5’和RNA的5’ end相接
Cut the RNA chain at a particular sequence of nucleotides by enzyme.
Then another enzyme adds poly-A tail, which is about hundreds of nucleotides long
3.non-coding sequence: about 80~10,000 nucleotides, or longer.
**Capping and spilicing take places before rna transcription completes.

12. Transcription (Procaryotes v.s Eucaryotes. Cont’d)
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.
3.mrna’s lifetime (mostly) depends on the untranslated sequence between 3’end of coding region to poly-A tail.
Lifetime of an mrna also determines the level of the protein. Generally, longer mrna lifetime leads to higher level of protein.
In modern biotech method, use eucaryotic ‘mrna’ for bacteria do not process rna anymore.

14. Translation (from RNA to Protein)
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.
4. Some differences caused by DNA in mitochondria which has transcription and protein synthesis system of its own.

15. 共有64種組合,真正有對應aa的只有61種,
UGA, UAA, UAG as terminating codon

16. Translation (tRNA with amino acid)
tRNA, transfer RNA
About 80 nucleotides long, with 3D structure.
Amino acid attaches to 3’ end.
1. Some region (4 parts) form double-helical structure with H bond.
2.T loop, D loop and anticodon loop
3.anticodon: 3 consecutive nucleotides complementary to mrna’s condon.

17. Translation (tRNA with amino acid. Cont’d)
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.
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.

18. Translation (tRNA with amino acid. Cont’d)
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...

19. Translation (Ribosomes for protein making)
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.
2. Large subunit:~49 proteins + 3 rrna
Small subunit:~33 protiens + 1 rrna; total 82 proteins + 4 rrna
3. Ribosomal protein are made in the cytoplasm and then sent in the nucleus.

20. If needed, two subunits come together on mRNA
If needed, two subunits come together on mRNA. After protein synthesis complete, they separate.
P site: 肽鏈最後一個aa所在
Large subunit: 肽鍵合成所在
Small subunit: mrna附著處

21. 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…

22. Translation (Condons for initiation)
Bacteria have no 5’ cap…
有特定的核甘酸序列可供核糖體附著
另原核可以在同一條mrna上出多個protein　但真核只能帶一個protein

23. Translation (Condons for terminal & others)
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.
4.5.　增近合成效率，蛋白質可以一邊合成一邊folding，可多個核糖體同時進行合成

24. PCR (Polymerase Chain Reaction)
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.mrna => to obtain cdna first by inverse transcription, then use cdna…
Knowledge of the DNA sequence to be amp is used to design two synthetic DNA oligo-nucleotieds

25. (1) 90ºC, open the DNA double-stranded 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!
5 min/cycle.
DNA初量約1~5ng即可

26. Reference. Essential Cell Biology, by Alberts et al., 1998, Garland
Some pictures are copied from wikipedia and

27. Thanks for your attention
THE END
Thanks for your attention