Lecture of nucleic_acids_[1] [autosaved]

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Transcript of Lecture of nucleic_acids_[1] [autosaved]

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Deoxyribonucleotide

Ribonucleotide

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Nucleic Acids: DNA and RNA• Nucleic acids are polymeric macromolecules (High molecular weight) or large 

biological macmolecules, essential for all known forms of life.• Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic

acid), are made from monomers known as nucleotides.• Each nucleotide has three components: a 5-carbon sugar, a phosphate group, and

a nitrogenous base. If the sugar is deoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA.

They are responsible for 2 fundamental properties: 1) Their ability to reproduce their kind or transfer genetic information 2) To undergo mutation-Nucleic acids are found in all living cells and viruses.- Friedrich Miescher in 1869 isolated what he called nuclein from the nuclei of

pus cells. Nuclein was shown to have acidic properties, hence it became called nucleic acid

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• Together with proteins, nucleic acids are the most important biological macromolecules; each is found in abundance in all living things,

• Where they function in encoding, transmitting and expressing genetic information.

• In other words, information is conveyed through the nucleic acid sequence, or• The order of nucleotides within a DNA or RNA molecule. • Strings of nucleotides strung together in a specific sequence are the

mechanism for storing and transmitting hereditary, OR genetic, information via protein synthesis.

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• In Eukaryotic cells, DNA occurs in combination with proteins called nucleoproteins chiefly histones of several types.

• Histones are small, highly basic proteins (known amino acid sequence)

• Genes of eukaryotes are present in chromatin which is made up of protein and DNA along with some RNA.

• Chromatin contains about equal amounts (by weight) of DNA and protein.

• Prokaryote (unicellular) with single chromosome without true nucleus.• Nuclear material scattered in cytoplasm, nucleoproteins found in

cytoplasm associated particularly with ribosome where RNA is ultimately concerned with protein synthesis.

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The nucleus contains the cell’s DNA (genome) RNA is synthesized in the nucleus and exported to the cytoplasm

Nucleus

CytoplasmDNA

RNA (mRNA)

Proteins

replication

transcription

translation

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Importance of Nucleic acids1) Nucleic acids are large molecules that carry tons of small details: all the genetic information. Nucleic acids are found in every living thing — plants, animals, bacteria, viruses, fungi — that uses and converts energy. Every single living thing has something in common.2) DNA is carrier of genetic information in all organisms with few exceptions

e.g. Tobacco mosaic virus, poliovirus, influenza virus and some bacteriophages use RNA for this purpose.3) Nucleic acids direct synthesis of proteins including enzymes.4) Mutations are essential for evolution of new varieties and species.5) All plant viruses have RNA while bacterial and animal viruses have either

DNA or RNA e.g Human immunodeficiency virus (HIV) and poliomyelitis are RNA viruses while vaccinia and Herpes are DNA viruses.

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6) Cancer research involves extensive studies of nucleic acids.7) Diseases like Gout and Orotic-aciduria are inborn errors of purine and pyrimidine metabolism respectively.8) It is possible to find out defective genes leading to serious diseases even before birth. 9) It is possible to synthesize genes and incorporate into genome of selected host cells.10) Genomic maps and libraries have been prepared.11) DNA from blood, seminal fluid can be analyzed by which parentage

and involvement of crimes like murder or raps can be detected with great precision. This is called DNA finger-printing.12) Gene therapy for certain genetic diseases in human beings is being

studied.

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Historical Summary of the Discovery of DNABy the 1950s, it was clear that DNA was the genetic material. The key

scientists who discovered and reported the structure of DNA were:

Linus Pauling 1901-1994

Robert Corey (1897-1971) Rosalind Franklin 1920-1958

Maurice Wilkins 1916-

James Watson 1928- Francis Crick 1916-2004

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DNA secondary structure – double helix

James Watson and Francis Crick, 1953- proposed a model for DNA structure

• DNA is the molecule of heredity (O.Avery, 1944)• X-ray diffraction (R.Franklin and M. Wilkins)• E. Chargaff (1940s) G = C and A = T in DNA

Francis Crick Jim Watson

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Watson-Crick model of DNA was based on X-ray diffraction picture of DNA fibres (Rosalind Franklin and Maurice Wilkins)

Rosalind Franklin

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Functions/Biochemical role of Nucleotides• Enter in structure of ATP• Form building blocks (Units) of Nucleic Acids (DNA & RNA)• Enter in structure of coenzymes (NAD, NADP, FMN, FAD: Hydrogen carriers),

acid carrier (Coenzyme-A), methyl donor (S-adenosyl methionine).• Enter in structure of cyclic AMP (cAMP, cGMP): 2nd messenger • Nucleoside 5'-triphosphates are carriers of energy. • Bases serve as recognition units. • Cyclic nucleotides are signal molecules and regulators of cellular metabolism

and reproduction. • ATP is central to energy metabolism. • GTP drives protein synthesis. • CTP drives lipid synthesis. • UTP drives carbohydrate metabolism.

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Nucleotide Function

– Energetic Intermediates– Adenine Enzyme Cofactors– Regulatory Molecules

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Nucleotides

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Phospho-Anhydrides and Phosphate Esters – High Energy Bonds

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Co-Enzyme A – Carrier for Acetyl units in intermediary

metabolism, fatty acid synthesis and oxidation

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NAD+/NADH, NADP+/NADPH and FAD/FADH2

• Redox Cofactors

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Regulatory/Signalling Molecules

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Nucleic Acids

DNA RNA

Central Dogma of Biology

DNA RNA Proteins Cellular Action

transcriptiontranslationDNAre

plic

atio

n(deoxyribonucleic acids) (ribonucleic acids)

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Two types of nucleic acid are found:• Deoxyribonucleic acid (DNA)• Ribonucleic acid (RNA) DNA is found in the nucleus with small amounts in mitochondria and

chloroplasts RNA is found throughout the cell Nucleic acids are polynucleotides Their building blocks are nucleotides

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DNA as genetic material: The circumstantial evidence

1. Present in all cells and virtually restricted to the nucleus2. The amount of DNA in somatic cells (body cells) of any given species is

constant (like the number of chromosomes)3. The DNA content of gametes (sex cells) is half that of somatic cells.

In cases of polyploidy (multiple sets of chromosomes), the DNA content increases by a proportional factor

4. The mutagenic effect of UV light peaks at 253.7nm. The peak for the absorption of UV light by DNA

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DNA contains two kinds of information:• The base sequences of genes that encode the amino acid sequences

of proteins and the nucleotide sequences of functional RNA (rRNA and tRNA).

• The gene regulatory networks that control the expression of protein-encoding (and functional RNA-encoding) genes.

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NUCLEOTIDE STRUCTUREPHOSPATE SUGAR

Ribose or Deoxyribose

NUCLEOTIDE

BASEPURINES PYRIMIDINES

Adenine (A)Guanine(G)

Cytocine (C)Thymine (T)Uracil (U)

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Garrett and Grisham, Biochemistry, Third Edition

(a) The pyrimidine ring system; by convention, atoms are numbered as indicated. (b) The purine ring system, atoms numbered as shown.

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Sugar “Pucker”

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The Building Blocks of DNA

-OO

H(OH)

HH

HHO

OP

O

O-

Purine orPyrimidineBase

PhosphatePentose sugar

Nucleoside

Nucleotide

1'

2'3'4'

5'-N-glycosidic bond

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Building Blocks–Nucleotides = Base + Sugar + Phosphate–Nucleosides = Base + Sugar–Nitrogen Bases

• Purines (5 + 6 membered rings) – numbering– Adenine Guanine

• Pyrimidines (6 membered ring) – numbering– Thymine Cytosine Uracil

–Pentose Sugars (numbering)• – Ribose• – Deoxy Ribose

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DNA and RNA nucleobases

NN

NNH

NH2

NNH

NNH

O

NH2

N

NH

NH2

O

H3CNH

NH

O

O

Guanine (G)Adenine (A)

Thymine (T)Cytosine (C)

NH

NH

O

O

Uracil (U)

NN

NNH

Purine

N

NH

Pyrimidine

12

34

5

6

3

2

16

4

57

8

9

(DNA only) (RNA only)

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Purine Nucleotides

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Pyrimidine Nucleotides

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nucleobase (Deoxy)nucleoside

5’-mononucleotide

Adenine (A)

Guanine (G)

Thymine (T)

Cytosine (C)

Uracil (U)

2’-Deoxyadenosine (dA)2’- Deoxyguanosine (dG)2’- Deoxythymidine (dT)2’- Deoxycytidine (dC)Uridine (U)

Deoxyadenosine 5’-monophosphate (5’-dAMP)Deoxyguanosine 5’-monophosphate (5’-dGMP)Deoxythymidine 5’-monophosphate (5’-dTMP)Deoxycytidine 5’-monophosphate (5’-dCMP)Uridine 5’-monophosphate (5’-UMP)

Nomenclature of Nucleobases, Nucleosides and Mononucleotides

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Structural differences between DNA and RNA

H3CNH

NH

O

O

Thymine (T)

NH

NH

O

O

Uracil (U)

DNA RNA

O

H

HHH

CH2

HO

HOBase

2'-deoxyribose

O

OH

HH

CH2

HO

HOBase

ribose

H

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Preferred conformations of nucleobases and sugars in DNA and RNA

HOO

OH

N

N

NH2

O

Anti conformation

HOO

OH

N

N

NH2

O

Syn conformation

HO

OH (OH)

HO

BASEHO

O

H (OH)

HOBASE

2' endo (B-DNA)

1'

3' endo (RNA)

3'

1'3'

2' 5'5'

7.0 A

5.9 A

Sugar puckers:

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Nucleosides must be converted to 5’-Triphosphates to be part of DNA and RNA

HOO

OH

OO

OH

PHO

HO

O

OO

OH

PO

PO

HOHO

OOH

Base Base

BaseOO

OH

PO

PO

OOH

BaseOH

OPO

HO

HO

Kinase

Kinase

Kinase

Monophosphate

DiphosphateTriphosphate

ATP

ATP

ATP

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Structure of Nucleic Acids

• Polymers of four nucleotides• Linked by alternating sugar-phosphate bonds• RNA: ribose and A, G, C, U• DNA: deoxyribose and A,G,C,T

nucleotide nucleotide nucleotide nucleotideP sugar

base

P sugar

base

P sugar

base

P sugar

base

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Phosphodiester bonds–Formed by Polymerase and Ligase activities–C-5' OH carries the phosphate in nucleotides–C5' - O - P - O - C3'–Phosphate pKa ~ 0–Natural Oligonucleotides have 5‘-P and 3‘-0H–Base hydrolysis due to ionizaiton of 2' OH in RNA

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The Structure of DNAAn antiparallel double helix

• Diameter of 2 nm • Circular in prokaryotic cells.• Length of 1.6 million nm (E. coli) • Compact and folded (E. coli cell is only 2000 nm long) • The linear eukaryotic DNA is wrapped around histone proteins to

form nucleosomes. • Base pairs: A-T, G-C

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DNA is arranged 5’ to 3’Connected by Phosphates

Linking in DNA biopolymer: DNA primary structure

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Oligonucleotide naming / drawing conventions

– 5’ - Left to Right - 3’ – pACGTOH – ACGT

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A, B and Z DNA• A form – favored by RNA• B form – Standard DNA

double helix under physiological conditions

• Z form – laboratory anomaly, – Left Handed– Requires Alt. GC– High Salt/ Charge

neutralization

A, B & Z DNA Kinemages

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•Polymers linked 3' to 5' by phosphodiester bridges. •Sequence is always read 5' to 3'.• In terms of genetic information, this corresponds to "N to C" in proteins.• The base sequence of a nucleic acid is its distinctive characteristic.• pGpApCpU, GpApCpUp, pGpApCpUp, GACU, dGACT

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Types of Nucleic acids• DNA - one type, one purpose: - a single DNA molecules in virus and bacteria- Eukaryotic cells have many diploid chromosomes mainly in nucleus,

but also mitochondria and chloroplasts.• RNA - 3 (or 4) types, 3 (or 4) purposes

– ribosomal RNA - the basis of structure and function of ribosomes – messenger RNA - carries the message– transfer RNA - carries the amino acids– Small nuclear RNA– Small non-coding RNAs

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DNA Backbone Flexibility

• Multiple Degrees of Rotational Freedom

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Semi-conservative Replication

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RNA: Typically single stranded: Produced during transcription• mRNA: Carries information encoded in genes to direct protein synthesis on ribosomes

– Derive from heterogeneous nuclear RNA (hnRNA)– RNA processed by splicing (removal of introns and joining of exons), capping (5’ end),

and polyA tail addition (3’ end)• rRNA: Components of ribosome: Protein synthesis

–Small subunit of ribosome: Single rRNA– Large subunit of ribosome: Large subunit rRNA, 5S rRNA, and in eukaryotes 5.8S

rRNA• tRNA: Carriers of activated amino acids used by ribosome for protein synthesis• snRNA: Small nuclear RNAs (Important in converting hnRNA to mature mRNA)• siRNAs: Small interfering RNAs: Degrade mRNAs (post-transcriptional gene silencing)• miRNAs: micro RNAs: Bind to mRNA and block translation • snoRNAs: Small nucleolar RNAs: Required for certain RNA modification

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Messenger RNAs

• Contain protein coding information– ATG start codon to UAA, UAG, UGA Stop Codon – A cistron is the unit of RNA that encodes one polypeptide chain– Prokaryotic mRNAs are poly-cistronic– Eukaryotic mRNAs are mono-cistronic

• Base pairing/3D structure is the exception– Can be used to regulate RNA stability termination, RNA editng, RNA

splicing

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Distinctive Base composition foretell base pairing patterns

– Hydrolysis of DNA and analysis of base composition• Same for different individuals of a given species• Same over time• Same in different tissues• %A = %T and %G = %C (Chargaff's Rules)

– Amino acid compositions vary under all three conditions

– No quantitative relationships in AA composition

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TelomeresTelomeres are sequences at the end of eukaryotic chromosomes that help stabilize the chromosome. Telomeres are repeats of the following sequence:

5’-(TxGy)n x and y = 1 to 43’-(AxCy)n The TG strand is longer

5’-TTTGGTTTGGTTTGGTTTGGTTTGGTTTGG…3’-AAACCAAACCAAACC…

Can be >10,000 nucleotides in mammals. The ends of the chromosome are replicated by the enzyme telomerase.