Transcription (전사) and Translation (번역)

Transcription of DNA into RNA

Basics of Transcription

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

Transcription of eukaryotes

Intron: 아무런 정보도 갖고 있지 않는 부위

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.

핵안에 있는 DNA의 유전정보를 세포질 안의 리보솜에 전달하는 RNA이다

핵 내에서 합성된 mRNA는 단백질 분자의 아미노산 배열을 지령하기 위해 핵에서 빠져나와 리보솜에 결합하고, 단백질 합성과정에서 아미노산 배열을 지령한다. 이렇게 DNA상의 유전정보를 전령하는 기능을 갖기 때문에 ‘전령RNA’라 부른다

mRNA

Translation of RNA into Protein

The Genetic Codes

The Genetic Codes

단백질 합성 시 상보적인 안티코돈을 가지고 있어 mRNA에 해당 아미노산을 운반해 주는 RNA이다.

tRNA의 구조 중 중요한 부분이 두 곳이 있는데, 하나는 tRNA의 맨 끝부분으로서 아미노산과 결합하는 곳이고, 다른 하나는 mRNA의 코돈에 상보적으로 대응하는 안티코돈이 있는 부분이다. mRNA의 코돈이나 tRNA의 안티코돈은 모두 세 개씩의 염기로 구성되어 있으며, DNA(디옥시리보뉴클레오티드)의 코드에 상보적으로 결합되는 부분이 mRNA의 코돈이고, 다시 mRNA의 코돈에 상보적으로 결합되는 부분이 tRNA의 안티코돈이다. 그러므로 DNA의 염기와 tRNA의 염기는 T(타이민) 대신에 U(유라실)만이 다르고 나머지는 모두 같다.

tRNA

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] 아미노산이 카르복실기를 통하여 다른 분자와 에스테르 결합을 하고 있는 것을 나타내는 연결형.

아미노아실아데닐산

아미노산과 nucleotide의 결합

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.

Large subunit

Small subunit

질문 1. 왜 16s 인가요?? 16s가 어떤 역할을 하는거죠?? 먼저 16s rRNA가 뭔지에 대해서 이야기 해야 겠네요... rRNA는 ribosome을 구성하는 RNA를 이야기합니다. 16s 라는 것은.. 그 RNA의 침강계수가 16이라는 것이죠... 16s rRNA는 리보좀을 구성하는 RNA이므로 잘 보전되어 있습니다. 하나의 primer 쌍을 통해 여러 균주의 16s rRNA를 증폭할 수 있는 것이 이 떄문입니다. 하지만 primer를 통해 증폭된 부분은 균 사이에 서로 다른 비교적 다양한 서열을 가지고 있습니다. 또한 예전부터 16s rRNA의 서열정보가 data화 되어 있으므로, 이를 비교해 본다면 쉽게 이를 이용할 경우, 균의 동정을 비교적 높은 신뢰도로 할 수 있게 되죠.. .

1. 어느 환경의 bacteria가 신종인지 알아보려면? 16S rRNA 해야되나요? 2. 흙에서 미생물을 무엇이 있는지 분석을 하려고 하면? 16S rRNA sequencing을 해야 하나요?

다수의 생물의 유전자 혹은 염기서열분석은 반드시 같은 부위(보통 유전자나 특정 서열)를 비교해야 근연관계를 알수 있지요.. 내 발가락과 옆사람 손가락, 그 옆사람 머리카락을 비교하며 누가 제일 긴 신체를 가졌고 누가 제일 굵은 몸통을 가졌다고 하면 안되겠지요? 모두 손가락만 혹은 머리카락만 비교해야겠지요? 그래서 이용되는 것이 16S rRNA 서열입니다. 이 서열은 진화과정에서 비교적 잘 보존이 되어 있고요 보존이 되어 있다는 말은 돌연변이가 덜 일어났다는 뜻입니다. 따라서 아직도 생물체들 간에 염기서열이 비슷하고요 비슷하면서도 진화과정 중에 축적된 돌연변이가 각 종을 충분하게 구분할 정도로 많다는 겁니다. 특히 이러한 copy수가 많은 유전자는 concerted evolution의 기작으로 한 생물체 혹은 한 종에서는 거의 서열이 바뀌지 않게 유지가 됩니다. 특히 16S rRNA 유전자 안에서도 거의 종간에 돌연변이가 없는 지역들이 있고요 이 부분을 primer로 만들어 PCR을 하면 어떠한 미생물 들의 16S rRNA 유전자도 다 증폭하여 서열분석을 할 수 있습니다. BLAAST를 하면 종 이름이 튀어나오게 되고요 (이미 기존에 등록된 종이라면) 아니라면 균을 분리하여 특성 구명을 해야겠지요? Multiple sequence alignment를 하면 생물체들간의 근연관계가 나오고요 따라서 가장 가까운 균들의 목록으로 추정한다면 무슨 균류인지도 확인 가능하고요..

첫 단계 (initiation): 작은 ribosomal subunit가 mRNA의 5’ end에 부착. fMet-tRNA가 mRNA의 초기화 codon에 plug-in됨. Larger ribosomal subunit가 자리에 안착되며, tRNA는 P site를 차지함 두 번째 단계 (elongation): A.A.가 부착된 두 번째의 tRNA가 A site로 이동하여, mRNA에 plug-in. peptide bond가 두 개의 A.A.간에 일어남. 동시에 첫 번째 A.A와 첫 번째의 tRNA의 결합은 깨짐. Ribosome은 mRNA chain의 5’ → 3’ 방향으로 이동. Repeat over and over. 세 번째 단계 (termination): Ribosome이 termination codon에 도달하면 polypeptide는 last tRNA와 분리되며, A site는 release factor가 차지하며, Release factor는 ribosome의 두 subunit를 분리한다.

단백질 합성의 3단계

첫 단계 (initiation): 작은 ribosomal subunit가 mRNA의 5’ end에 부착. fMet-tRNA가 mRNA의 초기화 codon에 plug-in됨. Larger ribosomal subunit가 자리에 안착되며, tRNA는 P site를 차지함

두 번째 단계 (elongation): A.A.가 부착된 두 번째의 tRNA가 A site로 이동하여, mRNA에 plug-in. peptide bond가 두 개의 A.A.간에 일어남. 동시에 첫 번째 A.A와 첫 번째의 tRNA의 결합은 깨짐. Ribosome은 mRNA chain의 5’ → 3’ 방향으로 이동. Repeat over and over.

5’ 3’

세 번째 단계 (termination): Ribosome이 termination codon에 도달하면 polypeptide는 last tRNA와 분리되며, A site는 release factor가 차지하며, Release factor는 ribosome의 두 subunit를 분리한다.

(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

1959년 노벨생리의학상

※ 프라이머: 특정 유전자 서열에 대하여 상보적인 짧은 단선의 유전자 서열 즉, oligonucleotide로 PCR진단, DNA sequencing 등에 이용할 목적으로 합성된 것임. DNA 중합효소에 의해 상보적인 유전자 서열이 합성될때 전체 유전자 서열 중에서 primer에서 부터 합성이 시작되는 기시절이 됨. 일반적으로 20∼30 base-pair의 길이로 합성하여 사용함

= Slowly proceeding strand

Need ligation

Proteins involved in DNA synthesis

The speed of a DNA polymerase: About 2,000 nucleotides/second Ex: E-coli 4.6x106 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

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 3x109 bases, mouse 2.5x109

fruit fly: 1.8x108, yeast 1.2x107

REPLICATION

DNA replication of prokaryotes: Non-linear Double-stranded cicular DNA Much smaller genome size than Eu: E-coli 4.6x106 bases Mycobacteria: 3x106 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

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Soil Microbial EcologySoil Microbial EcologySWS4303/5305SWS4303/5305

Bacterial Genetics

Definitions

• DNA: Deoxyribonucleic Acid• RNA: Ribonucleic acid• Transcription: Process of transcribing the p g

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 g– transcription

(DNA -->RNA)– translation

(RNA--> protein)

http://www.gene.com/ae/AB/GG/central.html

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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?

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Note double bonds

Note triple bonds

Note location of bases inside the double helix

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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)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 aGene: 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).

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• Most of the DNA in a bacterial cell is in a chromosome: large unit of DNA that is approximately 3,000 -6,000 kb.pp y , ,– How many genes?

DNA

mRNA

Transcription of template to message:

DNA mRNA

RNA polymerase

Start of gene End of gene

RibosomesProtein

Translation of the message to protein:

mRNA ProteinRibosomes

Plasmids

Extrachromosomal Elements

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Plasmid• Extrachromosomal circular genetic structure

that can reproduce autonomously but is usually not essential y– 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

•

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Horizontal Gene Exchange

Genes flow through soil!

Colonies in Soils are Mixed Species(The Great Melting Pot)

ConjugationTransformation

TransductionRecipient

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Conjugation

• First observed by Lederberg and Tatum in 1947.– Found that mixture of two strains of E. coli

produced offspring unlike either parent.Did ’ k h i– 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

-pilus

-

Attachment

Pilus

Plasmid Transfer

- disassembly

Channel formed

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Conjugation

Chromosome

Donor Recipient

Conjugation

Chromosome

Donor Recipient

Limits to Conjugation

• Cell:Cell contact• Metabolic state• Nature of the plasmid• Nature of the plasmid

– Transferable? Will it be maintained in recipient?

• Contrived system: Transfer rate at 40 days between 1 x 106 and 1.7 x 107 cells gram-1

day-1

– Focht et al., 1996

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Transduction

• Mediated by bacterial virus: bacteriophage• Phage makes mistake; takes some bacterial

DNA following infectionDNA following infection• Injects foreign DNA into genome of another

cell.• Can move entire plasmids in this way.

Generalized Transduction

attachmentDegradation Of chromosome

PackagingCell lysis

Release ofphage

Attachment

Barriers to Transduction

• Frequently species or strain specific due to attachment sites– Phage attaches to pilli; bacteria change thePhage attaches to pilli; bacteria change the

pillin– Phage can’t attach

• May become adsorbed to soil; can’t infect.• Host restriction modification systems.

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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 g– 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 i l di 2 4 5 T Mi ith h t t i iincluding 2,4,5-T. Mix with hosts containing 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.

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2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)

2,4-Dichlorophenoxyacetic acid(2,4-D)

CAM, TOL,

2,4,5-T,camphor,toluene,salicylate,chlorobenzoate

Nutrient feed

Chemostat

O2, inorganics

How do we make a 2,4,5-T degrader?

SAL, chlorobenzoate plasmids;bacteria from waste sites.

WasteContinue for 8-10 months…

http://www.studentsguide.in/microbiology/microbial-nutrition-growth/images/chemostat-continuous-culture-system.jpg

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B. cepacia AC1100

Chromosomes

IS931

Where did these genes come from???

2,4,5-T gene clusters

IS elements?

• Insertion sequence elements• “Jumping genes”• Can excise DNA and carry chunks from one• 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) ; f AB 340 kb d f EFGH 530 kb litftAB on 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.