How Cell Read the Genome

長庚大學中醫系

潘台龍博士

pan@mail.cgu.edu.tw

Goal of Human Genome Project

Introduction

DNA RNA Protein

translationtranscription

messenger RNA (mRNA)

genetic code (codon)

ribosome RNA (rRNA) amino acids-peptide bond-protein

transfer RNA (tRNA)

DNA and RNA polymerase

Deoxyribose nucleic acid (DNA) and ribonucleic acid (RNA)

• Pentose

Ribose-RNA

Deoxyribose-DNA

• Base

Purines Pyrimidines

Adenine (A) Uracil (U)

Guanine (G) Thymine (T)

Cytosine (C)

• PhosphateNu

cleo

tid

es

DNA is the Genetic Substance

Alfred Hershey & Martha Chase

35S & 32P

DNA

Double helix of two antiparallel chains with

complementary nucleotide sequences (1953

James D Watson and Francis H.C)

Structure of DNA

•Base-pair complementarity between a

larger purine (A & G) and a smaller

pyrimidine (C or T)

•Natural DNA: A with T by two hydrogen

bonds, G with C by three hydrogen bonds

Double helical DNADouble helical DNAStability of natural DNA

right-handed sugar-phosphate backbone-outside

bases-inside 0.34 nm between bases B form of DNA in most of the time in cells = a complete 3600 every 3.4 nm (10 bases) A form of DNA in non-aquwous solution = a coplete 3600 in 2.3 nm (11 bases)

Z (zigzag) DNA left-handed

Bent DNA Flexible about long axis Bent by/without DNA binding protein

Denaturation of DNADenaturation of DNA Melting tempreature (Tm) =separation of DNA double strands

Increased Tm =G-C rich DNA (3 hydrogen bonds) As DNA denatures, UV absorption increases.

Other factors to destabilize the double helix low ion concentration

alkaline solution formamide

Renaturation of DNA By lowering the temperature or increasing the ion

concentration Other single strand DNA not related in sequence

never affect the renaturation of two complimentary strands of DNA.

Many DNA molecules in all bacteria and many virus are circular

Nick (one of the strands is cut) is necessary to unwind and separate two strands

(one for circular, one for linear).

Natural nicking upon DNA replication

Experimental cleave with deoxyribonuclease

4 rules of the synthesis of nucleic acids

DNA RNAtranscription

1. Copy of temprate: DNA synthesis from DNA temprate retroviruss: DNA synthesis from RNA temprate 2. Nucleic acid strand growth is in one direction : 5’-3’

3. Special enzymes called polymerase make RNA or DNA RNA synthesis by copying DNA by RNA polymerase DNA polymerase requires a primerto make DNA 4. Duplex DNA synthesis requires a special growing fork. Leading strand-5’ to 3’-in the direction of the fork lagging strand (Okazaki)-discontinuous-DNA ligase

RNA polymerase

replication

DNA replication

primer

Template strand3‘

3‘5‘

5‘

Replication bubble of DNA under EM

DNA Sequencing

•Like PCR, it utilizes DNA polymerase and thermal c

ycling

•Only reads the sequence of 1 strand of DNA using

1 primer

•Utilizes the Sanger dideoxy termination method

•Reaction generates a population of dye-terminated

DNA fragments

Cycle Sequencing

DTCS

DeoxyNucloetide

DideoxyNucloetide

DTCS Extension and Termination

Extension and Termination

•Simultaneous reactions terminate at different lengths

•Reactions generate multiple fragments of all sizes from 1 to 1000+ bases

Separation and Detection

•Fragments are separated by capillary gel electrophoresis

•Laser-induced fluorescence of dye terminators is sequentially read by the PMT sensor

Sequencing Results

www.ncbi.nlm.nih.gov

Background: Several studies have reported telomere and telomerase in a variety of human malignant tumor.

•Telomere, located at the end of eukaryotic chromosomes, is considered important in protecting and stabilizing the chromosomal ends.•Telomerase is a ribonucleoprotein polymerase that can compensate for telomere losses.•Telomerase activity is present in almost all carcinomas and can be detected in some pre-neoplasias and early stage cancers.

Overview of Telomere and Telomerase

Telomere dynamics and chromosomal

instability in human epithelial cancers

Action of Telomere and Telomerase

One Gene, One Outcome???

One Gene, One Protein???

What is Single Nucleotide Polymorphism ?

•Many of differences among people have a genetic basis - alterations in the DNA that change the way important proteins are made.•Sometimes the alterations involve a single base pair (the smallest building block of DNA) and are shared by many people. Such single base pair differences are called "single nucleotide polymorphisms", or SNPs. However, the majority of the SNPs do not produce physical changes in people with affected DNA. •Estimate ~ 15M SNPs in total throughout human genome (one SNP every 200 bases).

Genetics of Drug Efficacy and ToxicityPharmacogenomics

Genetics of Drug Response

Future Potential of Pharmacogenomics

RNA

Similar to DNA in chemical makeup

However, additional hydroxyl group at the 2’ position and thymine (T) in DNA is replaced by uracil (U).

More chemically labile than DNA (cleaved

into mononucleotides even by alkaline solution)

Structure and function of RNAStructure and function of RNA

2 dimensional structure stem-loop and hairpin

3 dimensional structure (pseudoknot) small RNA: transfer RNA (tRNA)

large RNA: ribosome RNA (rRNA)Function

carry out genetic expression and convert to proteins Flexible about long axis

folded domains-catalytic ability (cut RNA an chain) e.g. phosphotransferase (cleave and unite-”splicing”) Various function in RNA bound to protein: the largest is ribosomes ( small nuclear ribonucleoproteins (“snurps”)

Transcription of RNA

Eukaryotic primary RNA transcripts are processed to form functional mRNAs

DNA RNA Proteintranslationtranscription

•RNA processing Modification of primary RNA is necessary for mRNA to be functional and capable of being translated into protein.•5’ cap and 3’ poly-A polymerase •Exon and intron exon: amino-acid sequence coding segment intron: protein non-coding segment Removal of intron (splicing) is required to make functional mRNA•5’ and 3’ untranslated regions also exist

mRNA carries information from DNA in a three-letter genetic code (codon)

DNA RNA Protein

translationtranscription

• Triple code (codon) : 4x4x4=64• 61 codons encode 20 amino acids• synonymous and degenerate :

e.g. leucine, serine, and arginine. Each have six.• Initiator(AUG)• Terminator ( UAA, UGA,UAG)• Reading frame (from initiator to terminator)• Frame shift yields different polypeptides.

A.C.G.UA.C.G.T

Modification of RNA Transcription

5‘ methylated cap

7-methylguanylate

RNA processing

Evidence of Splicing

Regulation of RNA Transcription

High Through-put Screening

cDNA Microarray Platform

Gene Analysis by Bioformatic

Protein synthesis: the three roles of RNA in translation

DNA RNA Protein

translationtranscription

• Messenger RNA (tRNA) encodes the genetic information copied from DNA• Transfer RNA (tRNA) The amino acids specified by the sequence of an mRNA are each attached to specific tRNAs, then carried to and deposited at the growing end of a polypeptide chain • Ribosome RNA (rRNA) 1. attracts mRNA, catalyzes peptide-bond formation and binds a set of proteins to form ribosomes. 2. Ribosomes bound tRNAs can move along an mRNA to translate its encoded genetic information into protein.

Overview in Protein Synthesis

Structure of tRNA

How to work in tRNA ?

Introduction to Proteins

Biopolymers —

Nucleic acids: store and transmit the genetic information of

the cell

Protein: play an enormous variety of roles; transport and

storage, structural framework, enzyme, antibody etc.

Each type of cell —

Has several thousand kinds of proteins, multiplicity of their

function.

Amino Acid

•Structure of the α–amino acid

C C

O

ON — —

H

H H

R

— H

C C

O

ON — —

H

H H

R

—H＋ －

•Stereochemistry of the α–amino acid

Chiral — D and L form

All of the amino acids incorporated by organism into proteins are of the L form

D-amino acids do exist in nature and play important biochemical roles

Examples, γ-aminobutyric acid: neurotransmitter in brain; thyroxine: thyroid hormone

Peptides and peptide bond

•Peptides

Properties of Amino Acid Side Chains

•Class of α–amino acid

Aliphatic Amino Acids — H, CH3, CH(CH3)2, CH2CH(CH3)2 ,

CHCH3CH2CH3 .

Amino Acid with hydroxyl- or sulfur-containing side chains —

CH2OH, CH2SH, CHOHCH3, CH2CH2SCH3

Aromatic Amino Acids — CH2Ø, CH2ØOH, CH2

Cyclic Amino acids — N-CH2CH2CH2-C

Basic Amino Acids — CH2 , CH2CH2CH2CH2NH3+,

CH2CH2CH2NHC(NH2)2

Acidic Amino Acids and their Amides — CH2COOH,

CH2CH2COOH, CH2CONH2, CH2CH2CONH2

C C

O

ON — —

H

H H

R

— H

N

N

Cysteine

C COO-H3N+ — —

H

CH2

SH

C COO-H3N+ — —

H

CH2

S-

+ H+pKa = 8.3

C COO-H3N+ — —

H

CH2

SH

C COO-H3N+ — —

H

CH2

SH

1. The side chain can ionize at high pH

2. Oxidation can occur to form a disulfide bind

C COO-H3N+ — —

H

CH2

S

C COO-H3N+ — —

H

CH2

S+ 2H+ + 2e-

CystineCysteine

OR

Absorption spectra of the aromatic amino acid

Titration curves of amino acids with ionizing side chains

Principle of IsoElectric Focusing

pKa1 pKa2

pKa1

pKa2

pI=1/2(pKa1+pKa2)

Modified Amino Acids

C COO-H3N+ — —

H

CH2

O

PO3 2-

O-Phosphoserine

C COO-H2N — —

H

CH2CH2

CH

OH

4-hydroxyproline

C COO-H3N+ — —

H

CH2

CH2

C

CH2

H3N+

—H — OH

δ-hydroxylysine

C COO-H3N+ — —

H

CH2

CH

COO--OOC

α-carboxyglutamic acid

Same Genome

The Challenge of Proteomics

Complex Proteome(s)

•Multiple Proteins for Each Gene

•Varied and Fragile Nature of Protein

•Quantitative and Qualitative Changes of the Proteome

•Structural and Functional Proteomics Studies

Post-translational Modification of Proteins

•Acetylation•ADP-ribosylation•Methylation•Phosphorylation•Glycosylation•GPI-anchoring•Myristoylation•Palmitoylation•Prenylation•Ubiquitinylation•SUMO addition•Proteolysis

1000 1500 2000

Mass (m/z)

Proteomics: Experimental Approach2-D Electrophoresis

Image analysis and entry into database

Excise spot and In-gel digestion

Extract peptides and Mass analyze

Database search

Protein Array in Clinical Medicine

Protein Array in Tumor Diagnosis

Protein profiling of Yersinia pestis cultured at its two physiological temperatures. 10 μg of crude cytosolic extracts of Y. pestis grown at 26°C versus 37°C were analyzed on a strong anion exchange chip (SAX-2). Proteins expressed only at 37°C, selected for purification and identification, were the 14.9 kDa (boxed) and 78.8 kDa (inset) proteins. The 14.9 kDa protein was identified as antigen 4 and the 78.8 kDa protein as the catalase/peroxidase KatY protein. Thulasiraman V et al., Biotechniques 2001,

Protein biochips for differential profiling

The protein peak at M/Z 2111 identified by the algorithm as belonging to theoptimum discriminatory pattern is indicated by the arrow, Lancet, 2002, 359

Use of proteomic patterns in serum to identify ovarian cancer

Tissue Array

In vivo Molecular Image

A tracer scan reveals a late stage of multistep tumor progression in which a primary tumor has spread to a number of sites in the rib cage, yielding the metastases seen here as hot-spots on a blue background.

Functional Genomics Approach In Molecular Medicine

Reference

• 4th Molecular Cell Biology• Biochemistry (Mathews)