GENETICS & DNA PN KAMARIAH

Genetic Material :
Heredity unit in organism 1st discover by Mendel Characteristic : o Information must contain information to construct entire organism (blue print) o Transmission able to transmit accurately from generation to generation and cell to cell o Replication able to be accurately copied o Variation account for variation within each species and among different species Working subunit of DNA Each genes contain a particular set of instructions Code for a particular protein or a particular function

Genes :

1940s scientist knew that chromosomes is the genetic material but; Chromosomes = DNA + RNA + histone protein Which one is genetic materials?

1st proposal Protein as the genetic materials

Because protein are large molecules with various structure and functions Little was known about nucleic acids The physical and chemical properties of DNA are too uniform to account for multitude (massive amount) of inherited traits

DNA as the genetic materials

A. Indirect evidence DNA is found on any chromosomes (in nucleus) only but more RNA and proteins found in cytoplasm Somatic cell of diploid organism contain twice the amount of DNA in germ cells Content of DNA molecules are same in different cell of an organism but content of RNA and proteins are different B. Direct evidence 1. Frederick Griffith Experiment (1928) Materials : 2 strains of Strptococcus Pneumoniae

Strain S (capsulated virulent *pathogenic*) Strain R (non-capsulated nonvirulent *non-pathogenic*) Observation : o Exp 1 : He injected mice with the S strain and the mice died o Exp 2 : He injected mice with the R strain and the mice survived o Exp 3 : He heat killed the S strain, S strains capsule still present and then injected the mice with dead S strain and the mice lived o Exp 4 : He injected dead S strain and live R strain into the mice and the mice died. He then detected the presence of live S strain bacteria with live R strain bacteria in the blood of the dead mice.

Hypothesis : Exp 1 : Strain S pathogenic Exp 2 : Strain R non-pathogenic Exp 3 : - capsule present but capsule not the factor cause pneumonia - No strain S found Exp 4 : - both live strain S and strain R found in mice blood - Harmless R strain cell has been transformed by materials from dead strain S cell - decendants of the transformed cell are pathogenic

In Griffith's experiment, after the S strain was heat killed, its DNA survived and was taken up by R strain. The S strain bacteria DNA enabled the R strain bacteria to grow a protective capsule, gain virulent properties and defeat the host's immune system Phenomenon of transformation occur but what is the transforming agent? 2. Avery, MacLeod & MC Carthy Experiment

Objective : to search for transforming agent Method : - use crushes S strain cell and extract its chemical components - Components separated and mixed with life R strain

3. Hershey & Chase Experiment (1952) Study T2 virus Bacteriophages labelled with : o Radioactive Sulphur (35S) for protein synthesis o Radioactive Phosporus (32P) for DNA synthesis Allowed labelled virus to infect bacteria Question : where are the radioactive after infection? Observation : o Radioactive Sulphur (35S) found in the capsule outside the bacteria. o Radioactive Phosporus (32P) found inside the bacteria

DNA & RNA

Both are genetic materials of the organism Known as nucleic acids RNA : genetic materials of some viruses (tobacco mosaic virus TMU) DNA : very large macromolecules o Building block of DNA and RNA nucleotides o DNA/RNA formed by the covalent linkage o 2 strands of DNA automatically formed double helix by H bond o RNA single strand o DNA associated with an array of different proteins to form chromosomes o A genome is the complete genetic materials of the organism Gene : 1) Exon (coding region for translate protein) 2) Intron (non-coding region for differentiation individual) The double helix structure of DNA was proposed by Watson and Crick in 1953 Based on discoveries of scientist : 1) Chargaff 2) Rosalin Franklins work

Chargaff Analyzed DNA composition of different organism Discovery : o DNA composition is species-specific ; amount and ratios of nitrogenous bases vary from one species to another o In every species studied, the base ratios was regular i.e. the number of A = T and G = C Double Helix DNA Structure (Watson and Crick) Consist of 2 strands of polynucleotide chains coiled together to form spiral (double helix) Found in opposite direction (anti parallel) and linked together by H bond Sugar and phosphate from the backbone outside the helix Nitrogenous base inside helix Purine bases pairs with Pyrimidine bases ; A bonded with T (double H bond) and G bonded with C (triple H bond) DNA molecules stabilized by : H bond between paired bases (collectively strong) Van der Waals forces between stacked bases DNA helix has uniform width 2nm Adjacent base pair are 0.3m apart Helix makes one full turn for every 3.4nm (10 or more nucleotides)

Base-Pairing Rules 1. Explain Chargaff findings o In every species studied, the base ratios was regular i.e. the number of A = T and G = C. o Thus in given DNA molecules : Since A must pair with T number of A = T Since G must pair with C number of G = C 2. Maintain consistent separation between 2 strands of DNA double helix Purines (2 ring base) Adenine Guanine Pyrimidines ( 1 ring base) Thymine Cytosine

3. Provide stability of DNA double helix i.e. : a. H bonds between bases b. Van der Waals forces between stacked bases. 4. The sequences of bases is highly variable along the length of DNA strand suitable for coding genetic informations 5. Suggest the general mechanism for DNA replication o Since the base pairing are specific; information on one strand complements the information along genes

The Central Dogma Concept A flow of genetic informations within organism. Genetic materials (DNA) : Must be able to store genetic information and transmit it accurately from generation to generation through Replication Must be able to control the phenotype development of an organism (genetic expression) through protein synthesis (Transcription and Translation)

Transcription

Translation

DNA

RNA
Nucleus Cytoplasm

Protein

Central Dogma

DNA REPLICATION
A process which a DNA molecule can produce an exact copy of itself 3 models of mechanism of DNA replication are proposed : o Semi-Conservative o Conservative o Dispersive

a) Semi-Conservative o 2 parental DNA strands separate o Each strand serves as template for the synthesis of a new DNA strands o The result is two DNA double helixes, which consist of one parental strand and one new strand (half conserve) b) Conservative o The two parental DNA strands join back together after replication o One daughter molecule contains both parental DNA strands (completely conserve) o The other daughter strand contains new DNA strands c) Dispersive o The parental double helix is broken into double stranded DNA segments that acts as templates for the synthesis of new helix molecules o The segment of parental and daughter (new) DNA are interspread in both with segments after replication

Meselson and Stahl (1958) Revised experimental approach to distinguish the 3 mechanism Proved that replication is semi conservative Use E.Coli that grown in 15N medium for new generations Then transfer into 14N medium Separate each generation (after 1 round of replication) analysed using Density Gradient Configuration.

DNA Replication Based on semi-conservative model; DNA Helicase, breaks the Hydrogen bonds between the bases (A-T, G-C) Double helix DNA unwind and open up to form replication fork DNA synthesis occurs in 53 directions, both DNA strands become template Both DNA strands are replicated simultaneously produced : o Leading strands : continuous synthesis 53 direction towards the replication fork o Lagging strands : discontinuous synthesis 53 direction against the replication fork

Formation of Leading Strands 1. RNA Polymerase (RNA primase) attach on template DNA 2. Produce RNA Primer short length RNA 3. RNA Polymer allows DNA Polymerase III to bind nucleosides complementary (1 sugar + 1 base + 3 phosphate group) to the template and replication begins 4. Replication in 53 direction 5. DNA Polymerase III removed

DNA Polymerase III : Elongated the strands by adding new nucleosides One by one nucleosides is added according to base-pairing rules (A-T, G-C) Catalyze the formation of phosphodiester bond (type of covalent bond) between the nucleotides of new strands For example : Base sequence DNA template B : 3 GGACGAACCAATAAG 5 Base sequence DNA new strand : 5 CCTGCTTGGTTATTC 3 The new strand is complementary to the template (old DNA strands)

Formation of Lagging Strands 1. RNA Polymerase attached to the DNA template at 3 end (near replication fork) 2. Synthesis RNA Primer on the antiparallel template strand (opposite direction of replication fork) 3. DNA Polymerase III binds to template and begin replication 4. Nucleosides join together by phosphodiester bond catalysed by DNA Polymerase III 5. Form short length DNA called Okazaki Fragments 6. Each fragments has RNA Primer 7. Freed from Okazaki Fragments by DNA Polymerase I 8. Okazaki Fragments are ligated (joined) together by DNA Ligase, forming continuous strand 9. Replication complete : a. Two double strand daughter DNA molecules formed b. Each with one parent strand and one new strand 10. The new double strand daughter twisted to form double helix DNA molecules

*Topoisomer : - To make sure double helix not distorted when it straighten - To manage double helix

PROTEIN SYNTHESIS
Protein structural & metabolic o eg: collagen, haemoglobin, actin, myosin, enzyme, antibodies etc o amino acids arranged in specific sequences based on gene o determines the physical and chemical nature of proteins

Archibalds Garrod (1908) medical doctor 1st to propose relationship between genes and production on enzyme (genes encode enzyme) Studied inherited disease Alkaptonin Normal individuals produce enzyme homogentisic acid oxidase that break down alkapton (chemical) Patients that lack the enzyme alkapton released into urine Urine change from yellow to brown and black when exposed to air In later life the patients will develop arthritis The disease is autosomal recessive inheritance Postulated that mutation in the gene caused the enzyme not produced Alkapton is not metabolism

Beadle and Tatom (1940) Studied bread mold, Neurospora crassa (exposed to X-ray) Mutants are defect in metabolic pathway that synthesizes amino acid arginine Mutants are defective in a single gene lack single enzyme Single gene control the synthesis of single enzyme One gene one enzyme hypothesis Wild Type Precursor Class I mutant Precursor Class II mutant Precursor Class III mutant Precursor

Ornithine

Citrulline

Arginine

Ornithine

Citrulline

Arginine

Ornithine

Citrulline

Arginine

Ornithine

Citrulline

Arginine

Gene A

Gene B

Gene C

Modification of hypothesis : o Enzyme are one category of proteins o All proteins are encoded by genes o Many proteins are constructed from 2 or more different polypeptides and each polypeptides is specified its own gene o Gene concept : one gene one polypeptide

Flow of Genetic Information : DNA controls cell activities control various types of proteins DNA : nucleotides sequences
controls

Protein : amino acid sequences

need

3 base for 1 amino acid

Molecular Gene Expression 1. Transcription (occur in nucleus) o Produces RNA copy of the gene o Information in DNA transcribed (copied) to mRNA molecule o DNA strand act as template o mRNA transcribed complementary to DNA template strand o mRNA carries information from DNA to ribosomes 2. Translation (occur in cytoplasm) o Synthesizing specific polynucleotides on a ribosomes o Genetic information on the mRNA is translated by ribosomes into amino acids sequence of polynucleotides o Require tRNA to carry a specific amino acid and add to growing polypeptide chain according to the code in mRNA * Transcription and Translation is called Central Dogma of gene expression Protein Synthesis in Living Things 1. Prokaryotes o No nucleus to separate transcription and translation o Translation can begin immediately while mRNA is being transcribed o Both occur in cytoplasm

2. Eukaryotes o Transcription in nucleus, translation in cytoplasm o Pre-mRNA produced (long, need to be process) then processed to produce mature mRNA o Mature mRNA exits nucleus into the cytoplasm to be translated

TRANSCRIPTION
A protein-coding gene codes the synthesis of a specific protein consist of : Promoter (initator): a base-pair sequence where transcription begins RNA-coding sequence : a base-pair sequence that include coding information for polypeptide chain specified by gene Terminator : a base-pair sequence that specifies the end of the mRNA transcription Evidence that RNA is intermediate molecule in protein synthesis i. DNA found in nucleus but protein found in cytoplasm ii. RNA synthesis in the nucleus (transcription) and chemically similar to DNA iii. RNA migrates to cytoplasm where protein synthesis (translation) occur iv. Amount of RNA is proportional to the amount of protein in cell

Transcription (How is mRNA produced?) Catalysed by the enzyme RNA Polymerase uncoil the protein-coding segment (a single gene) One strand become template (antisense strand) base sequence of mRNA is complementary to it The other strand (sense strand) has the same sequence as mRNA (except Thymine replaced by Uracil) protein-coding gene Activity of protein determine the structure and function of cell Protein link between genotypes and the phenotypes of organism 3 stages of Transcription : i. Initiation ii. Elongation iii. Termination Initiation o At the promoter site (start codon) o Specific recognition (identification) by RNA Polymerase of : - Promoter base sequence (prokaryotes) - A complex protein (eukaryotes)

i.

o RNA synthesis initiated addition of free RNA nucleotides by RNA Polymerase in the 5 3 direction o 3 5 strand will become a template for transcription process o Known as antisense strand (template) ii. Elongation o Adding of more free RNA nucleotides o Occur in 5 3 direction o mRNA strand is complementary to DNA template (antisense strand) o mRNA strand is same as the opposite DNA strand (sense strand) except Thymine (T) is replaced by Uracil (U) Termination o RNA Polymerase recognise the terminator base sequence o In prokaryotes, newly formed strand fold back to itself forming harspin structure o In eukaryotes, termination stop when termination factor (protein) is found on DNA strand o mRNA strand separate from DNA template

iii.

RNA Processing In eukaryotes only, genes contain Introns break up coding sequence Exons Introns; non-coding sequences (cannot be translated into proteins), transcribed to pre-mRNA but not translated Introns are removed and Exons are spliced (join) together to produce mature mRNA Mature mRNA are shorter than pre-mRNA because the Introns have been removed Splicing process process of removing Introns and join the Exons back together The mature mRNA released to cytoplasm

Genetic Code Information on mRNA in code form genetic code Triplet code 3 nucleotides in mRNA specify one amino acids in proteins (codons) Nonsense codons do not code for any amino acids (terminator codon) AUG start codon also for amino acids methionine UAA , UAG , UGA terminator codons (nonsense codon not code for any proteins)

TRANSLATION
Initiated by the assembly of mRNA, tRNA and ribosomal subunit (rRNA) Required various type of enzymes such as amino acids tRNA synthetase and ATP Transfer RNA (tRNA) o Transfer free amino acids from the cytoplasm and arrange them into polypeptide chain o Shortest RNA o Many parts are complementary to other part clover leaf shape o Contain anticodon that complimentary to codon on mRNA o Amino acids attach to 3 end of tRNA 3 stage of Translation : i. Initiation ii. Elongation iii. Termination Initiation o Initiaton factors protein attached to small subunit of rRNA o mRNA binds to small subunit o Initiators tRNA bind to mRNA and form functional complex o Initiator tRNA recognise the start codon AUG on 5 end of mRNA and binds to it o Large ribosomal subunit binds to small subunit o Initiator tRNA is located at the P-site o A-site is empty, ready to receive new charged tRNA with an amino acid code by the codon on mRNA Elongation 1. Charged (aminoacyl) tRNA carrying an amino acid enter A-site (use energy GTP) o Anticodon on tRNA is complementary to the codon on the mRNA o Initiator tRNA at the P-site o Amino acid met from initiator tRNA detached and bind with the new amino acid that at the A-site o Peptide bond is produced between the two amino acids 2. As the ribosome slide along the mRNA, the initiator tRNA will be removed from the complex through E-site (exits) and recycled o tRNA at A-site will occupy P-site and A-site is empty and ready to receive next tRNA o The process is repeated continuously until termination codon is detected

i.

ii.

The growing polypeptides is removed from tRNA at P-site and transferred to mRNA

3.

Peptide bond formed between amino acids at A-site and growing polypeptide at P-site o The formation of the peptide bond is called peptidyl transferase activity o Moves from 5 towards 3 end mRNA (every move is 1 codon) o Shifted tRNA at P-site to E-site and at A-site to P-site o A-site is empty, ready to accept next charged tRNA

iii.

Termination o Occur when a stop codon (UAA, UAG, UGA) is found at the A-site (no charged tRNA at this time) o Recognised by released factor that mimics the structure of tRNA o Released factor bind to stop codon o Ribosomal subunit mRNA and released factor dissociate

* Polyribosome (Polysome) o Translation can involve more than one ribosome o A group of ribosomes attached to single mRNA and translation occur simultaneously o Each ribosome will produced one polypeptide that is similar o Produced a number of polypeptides at a time

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