1. 生命科学发展简史
2. DNA
Nelson and Cox, Principles of Biochemistry
1. Deoxyribonucleotides and ribonucleotides
DNA或RNA是所有生命的遗传物质，其中只有部分病毒采用RNA作为遗传物质，所有的非病毒生物都采用DNA作为遗传物质。DNA和RNA为大分子（脱氧）核苷酸链，每个（脱氧）核苷酸都具有一个5‘磷酸基团、一个3’五碳糖、和一个碱基。
2. Phosphodiester linkages in covalent backbone
DNA或RNA链的延伸只能是从5‘到3’，因为DNA／RNA polymerase可以做用到DNA/RNA链的3‘OH基团上。The energy for polymerization come from dNTP (deoxynucleoside tri-phosphate). Thus the free dNTP needs to be hydrolyzed and attached to the free -OH which resides only in 3’. The other way around won’t be able to provide enough energy for the reaction.
3. Base pairing
A跟T配对，有两个氢健；G跟C配对，有三个氢健。为什么只能是这样的配对方式呢？Firstly, some options are ruled out because they can’t form more than 2 or 3 hydrogen bounds. However, G and T can form 2 hydrogen bonds and thus stable. G and T pairing can occur in RNA but not DNA because the DNA repair system will correct most of the Non-Watson-Crick pairing.
为什么DNA是双螺旋结构呢？碱基是疏水基团，而磷酸基是亲水基团，所以在DNA链中碱基在内部而磷酸基团在外部。DNA内部的碱基会一层一层地叠起来，再加上每个碱基的特定结构，最后DNA只有是双螺旋的时候才是最稳定的。
为什么DNA是反向互补呢？This is determined by the base parings. It’s pretty amazing that A-T and C-G paring both make the -OH expose in the opposite direction.
3. RNA
There are 5 major conserved RNAs in eukaryotes:
3.1. rRNA
rRNA is a component of ribosome and make up about 80% of total cellular RNA. Ribosome has ~60% rRNA and ~40% protein. Each ribosome has two subunits: large ribosomal subunit (LSU) and small ribosomal subunit (SSU).
Prokaryotes have 23S (~2.9kb) rRNA and 5S (~120nt) rRNA in LSU and 16S (1.5kb) rRNA in SSU. All the 3 bacterial rRNAs are usually co-transcribed as an operon. There is a internal space between 16S and 23S rRNA genes. Prokaryotic rRNA operon also contains tRNA.
Eukaryotes have 28S rRNA (4-5kb), 5.8S (150bp) rRNA, and 5S rRNA in LSU; and 18S (~1.9kb) rRNA in SSU. Eukaryotic rRNA genes are clustered in tandam repeats. Human has 5 rRNA gene clusters which contain 300-400 rRNA tandam repeats. Eukaryotic 5.8S, 18S and 28S rRNAs are also co-transcribed by RNA polymerase I, which is saperated b y 2 internal spaces. They are located in nucleolus organizer region on chromosomes. The processing of the pre-rRNA into mature rRNAs requires snoRNA. Eukaryotic 5S rRNA genes are in tandam arrays with ~200-300 copies in row and are transcribed by RNA polymerase III.5S rRNA clusters are located in nucleolus.
rRNA is present in all types of lifes. This extrodinary conserveness make rRNA one of the best sequences to do large scale phylogenetic analysis.
3.2. mRNA (~5%):
Transcribed by RNA polemerase II in eukaryotes nucleus. As soon as DNA starts to be transcribed, a RNA 7-methylguanosine (m7G) cap is added to the 5’ of pre-mRNA. 5’Cap is important in ribosome recognition and protection from RNases.
Imediately after the transcription is complete, ~250 adenosines will be added to the 3’.
Then, mature mRNAs will be transported from the nucleus to cytoplasm by mRNA export pathways and be translated. Most eukaryotic mRNAs are polysistronic (1 ORF) but some eukaryotic mRNAs as long as most prokaryotic mRNAs are polycictronic (several ORFs).
mRNAs in prokaryotes can survive 1-3 minutes on average, while mRNAs in eukaryotes can survive several minutes to days.
3.3. tRNA
A specific amino acid is attached to a tRNA. catalyzed by aminoacyl tRNA synthetase (tRNA ligase). tRNAs bearing amino acids are delivered to ribosomes between LSU and SSU by elongation factors.
Normally, eukaryotic species have several hundred of tRNA genes and also many tRNA derived psudogenes which are nonlonger functional (at least not as translation mediator)
tRNAs are considered to be the molecular fossils of RNA world. Primary tRNA-like molecules may have been existed very early in RNA world. Until tRNA evolved furthur to have function in carrying amino acid, RNA world turned into Ribonucleoprotein (RNP) world. As amino acid polymers (proteins) came into being, RNA could be used as template to be reverse transcribed into DNA, where DNA world got started.
In eukaryotes, tRNAs are transcribed by RNA polymerase III by recognizing the 2 highly conserved internal promoter sequences inside the tRNA genes: A box and B box. A box begins at +8 of mature tRNA and B box is within 30-60nt downstream of A box. Transcription of tRNAs terminates at 4 or more thymidines (T).
References:
1. A Role for tRNA Modificationsin Genome Structure and Codon Usage. Novoa et al., 2012 Cell
2. Recruitment of RNA polymerase III to its target promoters. Schramm and Hernandez 2002, Genes & Dev.
3. Diversity of tRNA genes in eukaryotes. Goodenbour and Pan., Nucleic Acids Research 2006
4. Aminoacyl-tRNA Synthesis., Ibba and Soll, Annual Review of Biochemistry 2000
5. Ribosomes and Translation., Green and Noller, Annual Review of Biochemistry 1997
6. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding. Dunkle et al., Science 2011
7. The Cryo-EM Structure of a Translation Initiation Complex from Escherichia coli. Allen et al., Cell 2005
3.4. small nuclear RNA (snRNA)
~150nt and transcribed by either RNA polymerase II or RNA polymerase III. The primary function of snRNA is pre-mRNA splicing mediated by splisosome. They are rich in Uridylate and generated in nucleus, thus called small nuclear RNA.
Splisosomes catalyze mRNA splicing in a extrordinarily precise way. Splisosome is a ribonucleoprotein (snRNP), consisting of snRNAs (U1, U2, U4, U5, U6) and over 150 proteins.
3.5. small nucleolar RNA (snoRNA)
~60-200nt. The primary function of snoRNA is to mediate modifications of other RNAs, mainly non-coding RNAs such as rRNAs, tRNAs, snRNAs. There are two major types of snoRNAs: the C/D box snoRNAs associated with RNA methylation, and the H/ACA box snoRNAs associated with pseudouridylation.
3.6. RNA secondary structure motifs (RNA folding):
Psedoknot, A-minor, Tetroloop, Ribose zipper, Kink-turn
3.7. Ribozymes:
1. self-splicing rRNA
2. RNase P: processing 5’ leader sequences of pre-tRNAs
3. Group I and Group II introns (self splicing) 
4. Protein
1. Amino acids:
Allison Fundamental Molecular Biology
2. Codon Bias:
3. Protein secondary structure, tertiary structure and protein folding
References:
1. Goldberg, A.L. 2003. Protein degradation and protection against misfolded or damaged proteins.Nature 426:895–899
2. Dobson, C.M. 2003. Protein folding and misfolding. Nature 426:884–890.
5. DNA replication
5.1. DNA polymerase
Allison Fundamental Molecular Biology
5.2 Bacterial DNA replication
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5.3. Eukaryotic DNA replication mechanism
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5.6. Rolling circle replication
5.7. Tolemere maintainence
5.4. DNA polymerase proofreading
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5.5. DNA repair and recombination
- Types of DNA damage
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DNA repair
Allison Fundamental Molecular Biology - DNA repair by homologous recombination in mammalian cells
- DNA repair by nonhomologous recombination in mammalian cells
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6. DNA Biotechnology
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- Restriction enzyme
- Clonning vectors
- Reporter genes
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7. Transcription in bacteria
7.1. RNA polymerase
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7.2. Transcription initiation, elongation, termination
8. Transcription in Eukaryotes
8.1 Overview
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