Transcription ( 전사 ) and Translation ( 번역 )

Transcription of DNA into RNA

Basics of Transcription

Transcription of eukaryotes Role of cap: To sign this is mrna to the cell Poly A tail: long string of adenine: to regulate stability of message. Without oly A tail, the message will be degraded rapidly.

Transcription of eukaryotes Intron: 아무런정보도갖고있지않는부위

Intron: codes that go out Exon: codes that stay Immature mrna of typical human gene: 30,000 bases Mature mrna of typical human gene: 1,500 bases

Alternative splicing One type of cell splices message to produce a certain protein, and other cell type splices the same gene to produce a different gene. Therefore, alternative splicing could create multiple proteins.

Ribosome - Large complexes of RNA and protein molecules. - Consists of two subunits, one small and the other large. - Large protein-synthesizing machine. - Has two sites involved in protein synthesis: a) A (aminoacyl) site, to which the incoming trna, with its amino acid, binds. b) P (peptidyl) site, to which the growing polypeptide chain is attached.

mrna 핵안에있는 DNA 의유전정보를세포질안의리보솜에전달하는 RNA 이다 핵내에서합성된 mrna 는단백질분자의아미노산배열을지령하기위해핵에서빠져나와리보솜에결합하고, 단백질합성과정에서아미노산배열을지령한다. 이렇게 DNA 상의유전정보를전령하는기능을갖기때문에 전령 RNA 라부른다

Translation of RNA into Protein

The Genetic Codes

trna 단백질합성시상보적인안티코돈을가지고있어 mrna 에해당아미노산을운반해주는 RNA 이다. trna 의구조중중요한부분이두곳이있는데, 하나는 trna 의맨끝부분으로서아미노산과결합하는곳이고, 다른하나는 mrna 의코돈에상보적으로대응하는안티코돈이있는부분이다. mrna 의코돈이나 trna 의안티코돈은모두세개씩의염기로구성되어있으며, DNA( 디옥시리보뉴클레오티드 ) 의코드에상보적으로결합되는부분이 mrna 의코돈이고, 다시 mrna 의코돈에상보적으로결합되는부분이 trna 의안티코돈이다. 그러므로 DNA 의염기와 trna 의염기는 T( 타이민 ) 대신에 U( 유라실 ) 만이다르고나머지는모두같다.

Transfer RNA - Transfer RNA molecules: is called the dictionary of the language of life translation of mrna into the language of proteins - More than 20 different trna in every cell, at least one for each of the 20 amino acids found in proteins. - Each trna has two important attachment sites: anticodon, 3 end of trna 1) anticodon: binds to the codon on an mrna molecule 2) 3 end of trna: attaches to a particular amino acid (enzyme: aminoacyl-tran synthetases). At least 20 different aminoacyl-tran synthetases. Aminoacyl trna: trna molecule that becomes covalently linked to an amino acid.

Structure of trna

Structure of Aminoacyl trna

아미노아실 [ aminoacyl] 아미노산이카르복실기를통하여다른분자와에스테르결합을하고있는것을나타내는연결형. 아미노아실아데닐산

아미노산과 nucleotide 의결합

Large subunit Small subunit

질문 1. 왜 16s 인가요?? 16s 가어떤역할을하는거죠?? 먼저 16s rrna 가뭔지에대해서이야기해야겠네요... rrna 는 ribosome 을구성하는 RNA 를이야기합니다. 16s 라는것은.. 그 RNA 의침강계수가 16 이라는것이죠... 16s rrna 는리보좀을구성하는 RNA 이므로잘보전되어있습니다. 하나의 primer 쌍을통해여러균주의 16s rrna 를증폭할수있는것이이떄문입니다. 하지만 primer 를통해증폭된부분은균사이에서로다른비교적다양한서열을가지고있습니다. 또한예전부터 16s rrna 의서열정보가 data 화되어있으므로, 이를비교해본다면쉽게이를이용할경우, 균의동정을비교적높은신뢰도로할수있게되죠...

1. 어느환경의 bacteria 가신종인지알아보려면? 16S rrna 해야되나요? 2. 흙에서미생물을무엇이있는지분석을하려고하면? 16S rrna sequencing 을해야하나요? 다수의생물의유전자혹은염기서열분석은반드시같은부위 ( 보통유전자나특정서열 ) 를비교해야근연관계를알수있지요.. 내발가락과옆사람손가락, 그옆사람머리카락을비교하며누가제일긴신체를가졌고누가제일굵은몸통을가졌다고하면안되겠지요? 모두손가락만혹은머리카락만비교해야겠지요? 그래서이용되는것이 16S rrna 서열입니다. 이서열은진화과정에서비교적잘보존이되어있고요보존이되어있다는말은돌연변이가덜일어났다는뜻입니다. 따라서아직도생물체들간에염기서열이비슷하고요비슷하면서도진화과정중에축적된돌연변이가각종을충분하게구분할정도로많다는겁니다. 특히이러한 copy 수가많은유전자는 concerted evolution 의기작으로한생물체혹은한종에서는거의서열이바뀌지않게유지가됩니다. 특히 16S rrna 유전자안에서도거의종간에돌연변이가없는지역들이있고요이부분을 primer 로만들어 PCR 을하면어떠한미생물들의 16S rrna 유전자도다증폭하여서열분석을할수있습니다. BLAAST 를하면종이름이튀어나오게되고요 ( 이미기존에등록된종이라면 ) 아니라면균을분리하여특성구명을해야겠지요? Multiple sequence alignment 를하면생물체들간의근연관계가나오고요따라서가장가까운균들의목록으로추정한다면무슨균류인지도확인가능하고요..

단백질합성의 3 단계 첫단계 (initiation): 작은 ribosomal subunit 가 mrna 의 5 end 에부착. fmet-trna 가 mrna 의초기화 codon 에 plug-in 됨. Larger ribosomal subunit 가자리에안착되며, trna 는 P site 를차지함 두번째단계 (elongation): A.A. 가부착된두번째의 trna 가 A site 로이동하여, mrna 에 plug-in. peptide bond 가두개의 A.A. 간에일어남. 동시에첫번째 A.A 와첫번째의 trna 의결합은깨짐. Ribosome 은 mrna chain 의 5 3 방향으로이동. Repeat over and over. 세번째단계 (termination): Ribosome 이 termination codon 에도달하면 polypeptide 는 last trna 와분리되며, A site 는 release factor 가차지하며, Release factor 는 ribosome 의두 subunit 를분리한다.

첫단계 (initiation): 작은 ribosomal subunit 가 mrna 의 5 end 에부착. fmet-trna 가 mrna 의초기화 codon 에 plug-in 됨. Larger ribosomal subunit 가자리에안착되며, trna 는 P site 를차지함

5 3 두번째단계 (elongation): A.A. 가부착된두번째의 trna 가 A site 로이동하여, mrna 에 plugin. peptide bond 가두개의 A.A. 간에일어남. 동시에첫번째 A.A 와첫번째의 trna 의결합은깨짐. Ribosome 은 mrna chain 의 5 3 방향으로이동. Repeat over and over.

세번째단계 (termination): Ribosome 이 termination codon 에도달하면 polypeptide 는 last trna 와분리되며, A site 는 release factor 가차지하며, Release factor 는 ribosome 의두 subunit 를분리한다.

( 세포질막, 원형질막 )

DNA replication

( 전사 ) ( 번역 )

Arthur Kornberg s in vitro (test tube) experiment using bacteria -DNA template - Nucleotides (dntps: datp, dctp, dgtp, dttp) - Short complementary primer - Enzyme 1959 년노벨생리의학상 프라이머 : 특정유전자서열에대하여상보적인짧은단선의유전자서열즉, oligonucleotide 로 PCR 진단, DNA sequencing 등에이용할목적으로합성된것임. DNA 중합효소에의해상보적인유전자서열이합성될때전체유전자서열중에서 primer 에서부터합성이시작되는기시절이됨. 일반적으로 20 30 base-pair 의길이로합성하여사용함

Need ligation = Slowly proceeding strand

Proteins involved in DNA synthesis

The speed of a DNA polymerase: About 2,000 nucleotides/second Ex: E-coli 4.6x10 6 base pair DNA replication time: 40 min

The kind of organisms: prokaryotes, eukaryotes, virus Eukaryotes: DNA living in nucleated cells Prokaryotes: DNA is not in a distinct nucleus Virus: DNA living in some kind of capsid Therefore, replication, transcription and translation are not entirely same REPLICATION DNA replication of Eukaryotes: Structure of your chromosomes: long linear chromosomes. Human chromosomes is a long double stranded molecule of DNA. You have 23 chromosomes, and together they make up three Billion of nucleotides of DNA. Genome size: human 3x10 9 bases, mouse 2.5x10 9 fruit fly: 1.8x10 8, yeast 1.2x10 7

DNA replication of prokaryotes: Non-linear Double-stranded cicular DNA Much smaller genome size than Eu: E-coli 4.6x10 6 bases Mycobacteria: 3x10 6 bases DNA replication of virus: Some virus have linear double, others circular, or linear single DNA. Some virus have only single strand RNA which creates DNA. How does it create DNA? Ans) by reverse transcriptase. Double stranded DNA of this virus can go into human chromosomes. Human can not get this out of genome. HIV = one of the retrovirus. Some of the important AIDs drugs are reverse transcriptase inhibitor

The Korea Times (2010. 11. 13)

DNA proofreading by DNA polymerase & exonuclease

DNA isolation PCR Gel electrophoresis DNA sequencing NCBI

DNA isolation kit

PCR instrument

After PCR, Agarose gel electrophoresis Materials 50X TAE (Tris acetate EDTA) Buffer (500 ml) - 121g Tris base in 250mL ddh2o - stir to dissolve - add 28.6mL acetic acid - add 50mL 0.5M EDTA ph 8.0 - measure in graduated cylinder and add ddh2o to 500mL 0.5X TAE Buffer (1,000mL) - take 10mL 50X TAE buffer - put into 990mL ddh2o in the bottle 1.5% agarose gel in TAE (200mL) - 3g agarose (Fisher, Agarose BP 1356-100 100gram, Lot 112638) - 200mL 0.5X TAE

Make reagents (50x TAE, 0.5x TAE, 1.5% agarose gel) (50x TAE buffer is for preparing 0.5x TAE buffer) Microwave 1.5% agarose gel in TAE for 2 minutes Put 2.5 µl of EthidiumBr into 1.5% agarose gel in TAE. Pour 1.5% agarose gel in TAE into Mupid cell Stand it for about 30 min for gel solidification Put the Mupid cell(5cm x 5cm) on the electrophoresis plate Load 5 µl of DNA marker (Exact Gene 1 kbp, PCR DNA ladder, Fisher) At first hole (left) in the cell Turn on Mupid electrophoresis Run 20 minutes

Mupid 전기영동장치

Gel image

Soil Microbial Ecology SWS4303/5305 Bacterial Genetics Definitions DNA: Deoxyribonucleic Acid RNA: Ribonucleic acid Transcription: Process of transcribing the blueprint in DNA to mrna; production of the message. Translation: translating the message to protein Gene: The functional and physical unit of heredity; segment of DNA that encodes a peptide. Genome: complement of genes in cell. MICROBIAL (Prokaryotic) GENETICS: assume basic knowledge of transcription (DNA -->RNA) translation (RNA--> protein) http://www.gene.com/ae/ab/gg/ central.html 1

DNA DNA (deoxyribonucleic acid) is the fundamental template which directs all processes in the cell. Double stranded polymer Phosphate:deoxyribose (sugar) backbone; two strands held together with H-bonding between bases (purines and pyrimidines). Purines: adenine (A) and guanine (G). Pyrimidines: cytosine (C) and thymine (T) Fundamental building blocks ( monomers ) of the DNA polymer are nucleotides. phosphate:sugar:base = nucleotide deoxyadenosine monophosphate (damp) deoxycytidine monophosphate (dcmp) deoxyguanosine monophosphate (dgmp) thymidine monophosphate (TMP) The two strands of the helix are partially held together by specific hydrogen bonds between A:T and G:C. A:T has two H-bonds; G:C has three H-bonds. What does this mean for relative stability? 2

Note double bonds Note triple bonds Note location of bases inside the double helix 3

DNA is Arranged in Chromosomes Most of the DNA in a bacterial cell is in a chromosome: large unit of DNA that is approximately 3,000-6,000 kb (3 million to 6 million base pairs). Base pair (bp) is the unit of size (number of monomers) All of the DNA required for the normal functioning of the cell are located on the chromosome. Most bacteria: one chromosome. Exceptions -Proteobacteria (some Pseudomonas, Rhizobium, Sphingomonas): 2-3 chromosomes. Most bacterial chromosomes are circular; exception: some high G+C gram positive bacteria (Streptomyces, Rhodococcus) have linear DNAs. These are actinomycetes. High G+C gram positives? DNA is typically supercoiled. Genes Genetic information in DNA is encoded in the sequence, or the specific order, of bases in a gene. The sequence of DNA is not random! Gene: segment of DNA involved in producing a polypeptide chain. The functional and physical unit of heredity; piece of DNA that encodes a peptide Three nucleotides encode a specific amino acid (the genetic code): codon A gene typically consists of 1000-2000 base pairs (bp), i.e. 1-2 kb (kilo basepairs). 4

Most of the DNA in a bacterial cell is in a chromosome: large unit of DNA that is approximately 3,000-6,000 kb. How many genes? DNA Start of gene RNA polymerase End of gene mrna Transcription of template to message: DNA mrna Protein Ribosomes Translation of the message to protein: Ribosomes mrna Protein Plasmids Extrachromosomal Elements 5

Plasmid Extrachromosomal circular genetic structure that can reproduce autonomously but is usually not essential 1-10% of the size of chromosome provides genetic diversity and enables bacteria to rapidly adapt to a wide range of ecological niches Some plasmid-encoded functions antibiotic and toxin production antibiotic and toxin resistance enzymes for xenobiotic degradation host-range genes (e.g. Rhizobium) dinitrogen fixation genes pigments 6

Horizontal Gene Exchange Genes flow through soil! Colonies in Soils are Mixed Species (The Great Melting Pot) Transformation Conjugation Recipient Transduction 7

Conjugation First observed by Lederberg and Tatum in 1947. Found that mixture of two strains of E. coli produced offspring unlike either parent. Didn t know mechanism. Conjugation: requires cell:cell contact. Usually plasmid mediated. Spread of plasmid encoded genes among group of bacteria. Most important of three genetic exchange mechanisms. Donor Pilus Recipient Plasmid Transfer pilus - Attachment Pilus - disassembly Channel formed 8

Conjugation Chromosome Donor Recipient Conjugation Chromosome Donor Recipient Limits to Conjugation Cell:Cell contact Metabolic state Nature of the plasmid Transferable? Will it be maintained in recipient? Contrived system: Transfer rate at 40 days between 1 x 10 6 and 1.7 x 10 7 cells gram -1 day -1 Focht et al., 1996 9

Transduction Mediated by bacterial virus: bacteriophage Phage makes mistake; takes some bacterial DNA following infection Injects foreign DNA into genome of another cell. Can move entire plasmids in this way. Generalized Transduction attachment Packaging Degradation Of chromosome Cell lysis Release of phage Attachment Barriers to Transduction Frequently species or strain specific due to attachment sites Phage attaches to pilli; bacteria change the pillin Phage can t attach May become adsorbed to soil; can t infect. Host restriction modification systems. 10

Transformation Uptake of naked extracellular DNA Incorporation into genome. Requirement of sequence similarity; due to homologous recombination Originated as method of obtaining nutrients DNA expensive to make Must overcome extra- and intra-cellular nucleases DNA short lived in soil. Extracellular DNA may be stabilized by adsorption to soil. Less important of three genetic exchange mechanisms Conclusion conjugation, transformation and transduction permit genetic exchanges among bacterial cells maintain genetic variability and thus ecological stability of bacterial populations Plasmid Assisted Molecular Breeding: Kellogg et al. (1981) Science 214:1133-1135 No single bacterial strain capable of growth on the herbicide 2,4,5-T. Isolate strains from soil from toxic waste site including 2,4,5-T. 245T Mix with hosts containing i many known catabolic plasmids (e.g. SAL, CAM, pac25(3- CB), TOL) Incubate in chemostat for several months, gradually weaning the system off substrates for above plasmids, and onto 2,4,5-T as sole source of carbon. Isolate strains capable of growth on 2,4,5-T as sole C- source. 11

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) 2,4-Dichlorophenoxyacetic acid (2,4-D) Nutrient feed 2,4,5-T, camphor, toluene, salicylate, chlorobenzoate How do we make a 2,4,5-T degrader? O 2, inorganics Chemostat CAM, TOL, SAL, chlorobenzoate plasmids; bacteria from waste sites. Continue for 8-10 months Waste http://www.studentsguide.in/microbiology/microbial-nutrition-growth/images/chemostat-continuous-culture-system.jpg 12

B. cepacia AC1100 Chromosomes IS931 2,4,5-T gene clusters Where did these genes come from??? IS elements? Insertion sequence elements Jumping genes Can excise DNA and carry chunks from one piece of DNA to another Plasmid to chromosome, chromosome to plasmid, plasmid to plasmid Plasmid Evolution Burkholderia cepacia AC1100 was isolated by growth on 2,4,5-T; where did this strain come from?? Five replicons present in AC1100 (150 kb to 4 Mb) ; tftab fabon 340 kb, and tftefgh on 530 kb replicon. IS931 upstream of the tft gene clusters. IS931 capable of moving intervening sequences. Foreign genes may have been recruited by IS elements. 13