Molecular Basis & Nucleic Acids: DNA and RNA

• Molecular basis focuses on DNA since it contains human body's genetic information
• RNA aids in transfer and expression of human body genetic information

Nucleic Acids

• Nucleic acids are polymers consisting of nucleotide chains
• Deoxyribonucleic acid (DNA) exists for the majority of organisms
• Ribonucleic acid (RNA) also acts as a genetic material in some viruses
• RNA mostly functions as a messenger, adapter, structural, and catalytic molecule

DNA Structure

• A long polymer of deoxyribonucleotides
• The length is defined by base pairs, which is characteristic of the organism
• Bacteriophage φX174 contains 5386 nucleotides
• Bacteriophage lambda contains 48502 base pairs
• Escherichia coli has 4.6 × 10bp
• Human DNA has 3.3 × 10bp

Polynucleotide Chain

• A nucleotide possesses three components:
  • A nitrogenous base
  • A pentose sugar
  • A phosphate group
• Purines like Adenine (A) and Guanine (G) are two types of nitrogenous bases
• Pyrimidines like Cytosine (C), Uracil (U) and Thymine (T) are also nitrogenous bases
• Cytosine is common to both DNA and RNA; thymine is present in DNA
• A nitrogenous base links to the OH of 1’ C pentose sugar through N-glycosidic linkage to the nucleoside
• A phosphate group links to OH of 5’ C of a nucleoside through a phosphoester linkage, forming a nucleotide
• Two nucleotides are linked through a 3’-5’ phosphodiester linkage, forming a dinucleotide; more nucleotides are joined to form a polynucleotide chain
• A polymer contains a free phosphate moiety at the 5’ end of the sugar (5’ end) and a free OH of 3’ C group at the other end (3’ end)
• The sugar and phosphates form the backbone; nitrogenous bases link to sugar moiety and project backbones
• RNA contains an additional –OH group at the 2’ position in ribose and has uracil in place of thymine

DNA Discovery

• Friedrich Meischer first identified acidic substance DNA in the nucleus (1869) that he named ‘Nuclein’
• James Watson and Francis Crick proposed the Double Helix model for DNA structure (1953), using X-ray diffraction data by Maurice Wilkins and Rosalind Franklin
• Erwin Chargaff observed that for double-stranded DNA, the ratios between A and T and G and C are constant and equal to one
• Each strand acts as a template for synthesising a new strand, producing identical daughter DNA molecules

Double-Helix Salient Features

• Two polynucleotide chains with a sugar-phosphate backbone and bases projecting inside
• The two chains run antiparallel, with one strand in a 5’→3’ polarity, the other in 3’→5’
• Bases pair through hydrogen bonds, A forms two bonds with T, G forms three bonds with C
• A purine always pairs opposite a pyrimidine, generates a uniform distance between the two strands
• The two chains are coiled in a right-handed fashion, with a helix pitch of 3.4 nm and roughly 10 bp per turn
• The distance between a bp = 0.34 nm, H-bonds provide stability
• Francis Crick proposed the Central Dogma to describe flow of genetic information: DNA → RNA → Protein
• In some viruses, the flow of information reverses (RNA to DNA)

DNA Helix Packaging

• Distance between two consecutive base pairs = 0.34 nm (0.34 × 10-9 m)
• DNA double helix length in a typical mammalian cell: 6.6 × 109 bp × 0.34 × 10-9 m/bp = approximately 2.2 meters
• DNA is packaged in a cell with an approximate dimension of 10-6 m

Packaging in Prokaryotes vs. Eukaryotes

• In prokaryotes (e.g., E. coli), DNA is held with proteins with positive charges in a region called 'nucleoid'
• In eukaryotes, the DNA is held with positively charged, basic proteins called histones
• Histones are rich in lysine and arginine amino acid residues
• Eight molecules of histones are organised to form a histone octamer
• Negatively charged DNA is wrapped around the positively charged histone octamer, forms a nucleosome
• A repeating unit in nucleus called chromatin, with ‘beads-on-string’ structure under electron microscope and contains 200 bp of DNA helix
• The beads-on-string structure are packaged to form chromatin fibres which are further coiled and condensed at metaphase of cell division to form chromosomes

Chromosomal Proteins

• Non-histone Chromosomal (NHC) proteins required for packaging of chromatin
• Euchromatin is loosely packed, stains light
• Heterochromatin more densely packed and stains darker
• Euchromatin is transcriptionally active, heterochromatin is inactive

Genetic Material Search:

• Friedrich Meischer discovered nuclein, Mendel proposed inheritance principles (around same time)
• It took a long time to discover and prove that DNA acts as a genetic material

Transforming Principle, Griffith

• Frederick Griffith experimented with Streptococcus pneumoniae (1928)
• He saw miraculous transformation in the bacteria since they changed physical form
• S strain bacteria have a mucous coat, while R strain bacteria do not
• Mice injected with S strain (virulent) die, but mice infected with R strain do not develop pneumonia
• Griffith concluded that R strain bacteria had been transformed by the heat-killed S strain bacteria
• A 'transforming principle' from the heat-killed S strain enabled the R strain to synthesise a mucous coat and become virulent
• The experiments did not yield the biochemical nature of genetic material

Biochemical Characterisation of Transforming Principle

• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-44) determined the biochemical nature of the transforming principle
• Genetic material thought to be protein
• DNA alone transforms bacteria, protein-digesting enzymes and RNA digesting enzymes do not affect transformation
• Digestion with DNase inhibits transformation, suggesting that DNA = the hereditary material

The Genetic Material is DNA

• Alfred Hershey and Martha Chase (1952) gave unequivocal proof that DNA is the genetic material
• They worked with bacteriophages (viruses infecting bacteria)
• Viruses grown on radioactive phosphorus contained radioactive DNA, but not protein
• Viruses grown on radioactive sulfur contained radioactive protein, but not DNA
• Bacteria infected with viruses that had radioactive DNA were radioactive, DNA entered from virus to bacteria
• Bacteria infected with viruses that had radioactive proteins were not radioactive, showed proteins did not enter
• DNA is passed from virus to bacteria = genetic material

Genetic Material (DNA versus RNA)

• Hershey-Chase experiment resolved debate between proteins/ DNA, DNA is genetic material
• RNA acts as a genetic material in some viruses, for example, Tobacco Mosaic viruses, QB bacteriophage
• Chemical structures of DNA and RNA affect genetic materials

Genetic Material Criteria:

• Molecule: must generate its replica, be chemically and structurally stable, provide scope for slow changes (mutation), express itself (Mendelian Characters)
• Rule of base pairing makes them able to direct duplications; proteins fail to fulfill first criteria by lacking that trait
• Evident in Griffith's 'transforming principle,’ heat killed bacteria but did not destroy some of the properties of genetic material
• DNA’s complementary strands will come back together if appropriate conditions are provided
• Stability: 2'-OH group present at every nucleotide in RNA is a reactive group, so DNA is less reactive and structurally more stable
• Thymine adds additional stability to DNA unlike Uracil
• DNA/RNA mutate, with RNA mutating faster since it is unstable because viruses mutate faster
• RNA can directly code for protein synthesis, but DNA depends on RNA for it

RNA World

• Evolved around RNA and was likely first genetic material
• Ribozymes act as genetic material and catalysts
• DNA evolved from RNA with mods for stability like double strands and editing

Replication Proposed Scheme

• Watson and Crick proposed the scheme, with strands acting as templates to synthesize complementary ones
• After, molecule would have one parental and the new strand, now known as semiconservative DNA Replication

Replication Machinery and Enzymes

• Enzymes, DNA-dependent DNA polymerase (or main enzyme) templates DNA to catalyse creation of deoxynucleotides
• 4.6 multiplied by 10 to the 6th E. coli (compare to human complete replication in the 18 mins (2000bp per second)
• Deoxyribonucleoside triphosphates serve two purposes: substrates + energy for polymerisation
• Long NA molecules cannot split entirely due to energy constraints, so replication occurs in replication fork
• Replicates 5 prime to 3 prime which causes difficulties, can be continuous or discontinuous (depending on template)
• Discontinuous fragments are joined by DNA ligase as DNA polymerase can’t start the process.
• A region in E. coli DNA dictates the Origin of Replication where a vector will be needed for a piece of DNA to propagate during Recombinant DNA procedures

Transcription

• Process of DNA info being copied into RNA, template is needed to synthesise complementary RNA
• Is limited to the sequence and one strand, defining these boundaries is needed since DNA is large
• No use to copy both since RNA would have to translate into different proteins which complicates genetic info too much
• RNA and DNA would become double-strainded, which is futile and prevents translation

Transcription Unit

• A Promoter: DNA sequence where RNA polymerase binds to start transcription.
• The Structural Gene: DNA sequence that is transcribed into RNA.
• A Terminator: DNA sequence that signals RNA polymerase to stop transcription.
• DNA-dependent RNA polymerase catalyses polymerisation of one way with 3-5 primer polarity acts as a template strand
• Coding Strand: 5’→3’ (does not code for anything but same as RNA)
• The promoter and terminator marks the structural gene, where DNA determines the coding RNA’s bind site and strands

Transcription Unit and Gene

• Gene: inheritance location, encodes tRNA, rRNA, segment (cistron) that is polypeptide encoding or transcription unit
• Structural gene: monocistronic (eukaryotes) or polycistronic (mostly in bacteria / prokaryotes) which are often interrupted with coding segments called exons
• Introns: sequences not transferred in mature processing (split-gene)
• Genes need regulatory sequence as they are also inheritance so a gene may not always contain RNA/proteins

Types of RNA and Transcription

• Three types of RNAs are needed in a cell to create a protein: messenger/mRNA, transfer/tRNA, and ribosomal/rRNA
• MRNA provides the template, TRNA holds the amino acids and reads code, rRNA is translation for structural and catalytic roles
• Bacteria uses a single DNA-dependent RNA polymerase that catalyses transcription of ALL types of RNA
• Polymerase initiates and binds transcription, using nucleoside triphosphates as a substrate and using Sigma factor proteins to elongate
• Transcript: primary product that has introns AND exons but is non-functional
• Splicing: introns are removed, exons are in defined order and spliced with additional processing like capping and tailing
• Capping: methyl guanosine triphosphate end is added to 5’ of hnRNA and tailing adds 200-300 adenylate residues at the 3’ end
• MRNA is transported outside the cell, that represents the ancient feature of the genome since split-genes may represent introns and splicing leads to RNA dominance
• There is now more understanding and assumed importance in RNA

Genetic Code

• Replication/transcription forms nucleic acid that copies the other to make new nucleic acid; the process in terms of complimentation
• Protein needs to transfer genetic information to transfer nucleotide polymers to synthesise Amino Acids, since nucleotides and AAs don’t exist theoretically
• If biochem codes (Genetic material) codes and determines and deciphers the DNA codes it will be challenging
• George Gamow said if 4 molecules codes for 20 amino acids, codes needs to be based on combo and that 3 nucleotides are needed for 20 AAs (triplet form makes 64 codons)
• Proof chemical was through Har Gobind Khorana which was instrumental in RNA synthesis.
• Marshall Nirenberg’s cell-system for protein helps the code be deciphered and Severo Ochoa uses enzyme RNA polymerase + Enzymatic RNA to make a checkerboard guide

Salient Genetic Codes

• 61 triplets of the codons codes for amino acids and other three are for stopping the coding (end process)
• degenerate: AA are coded more than one and not more than one codon, meaning the code can be manipulated
• Codon is in MRNA, no punctuation and there are no punctuations
• Universal codes: from bacteria to human
• AUG: dual functions that code for methionine + Initiation
• Stop signals: UAA, UAG, UGA

Mutation and Genetic Code

• Mutations (deleting / insertions of DNA affect the reading frame from points of insertion)
• Framshift Mutation: deletions do not alter coding

TRNA - Adapter Molecule

• CRICK proposed code needs mechanism with structure and speciality AA that would bind and read and recognise amino acids and tRNA/sRNA
• TRNA contains anticodon loop with complimentary codes to AA acceptor to bind Amino Acids
• tRNA’s role in initiation is through initiator tRNA

Translation

• Process for polymerising AA from polypeptide + AA sequences define by bases in mRNA and joined through a peptide (energy is needed to make)
• AA are activated in the ATP presence + cognate TRNA that are specific. if 2 charged tRNA are nearby then peptide bonds start between them
• Ribosomes would reduce energy for it all with catalysts made from enzyme ribosome to create the formation of peptide bond
• Translational Unit: RNA sequences that contains start and stop in Polypeptide and those regions are where they lack translation, that requires efficiency + regulation

Regulation of Gene Expression

• Concerns levels to express in formation of Polypeptide + Eukaryotes that transcriptionally form in primary transcript
• Processing depends on splicing, nucleus uses cytoplasm and translation
• Cells uses genes form different funciton based on metabolism and condition where expression regulate
• Prokaryotes: transcriptional initiations regulate and start access, regulatory proteins can be activators that change + and repressors for -
• Regulator interaction can switch off sequences names OPERATORS

LAC Operon

• Jacob and Monod where the first to regulated system which is polycistronic
• Has only 1 regulatory (I gene) + Structure (L,Y,A) codes lactose I gene codes repressor
• Z gene creates beta to hydrolyse and Y increases permease and permeability while An encodes transacetylase: all for lactose metabolism
• LActose is the beta substrate as the inducer regulates, without carbon the lactose is transported. low amounts help permeate cell/ lactose can enter

Human Genome Project

• Goal of figuring out human DNA sequence, can isolate DNA through simple techniques.
• Called MEGA cause aims were big, estimate cost to be 9 billion
• 3300 would store info and specialized to for retrieval as bioinformatics has new area
• HGP had important goals to know identify the approximate numbers of genes to store + other sectors and ethical sectors
• Project co-ordinated by US department and wellcoem with additional contributions from germany and china completing in 2003 since dna variations lead to evolutionary methods
• Method: tag all genes to coding and annotate + converted Random fragment + clone host
• SEQUENCERS that automated frager and linked to each chromosome that lead to the next sequence with polymorph Endonuclease where application of repeats is on fingerprints
• Humans genes: bases genes, vary greatly, estimated at levels less than 2, genomes, repeated sequences and identified around 1.4 locations
• App + Chall: find know of research with creativity of understanding where HG sequence can leads to study of questions

DNA Fingerprinting

• It needs to isolate genetic differences in populations that shows polyorphic Appearance that compare the sequences through repeating base pair
• Can classify satellite dna with segment to form polymorphs and tissue to show forensics as inherited patterns to create DNA fingerprints for testing and mapping + genetic testing and it is a tool due to mutations, polymorphisms

Biology Summary

• Polymorphism - Genetic that causes individuals ability impaired to function, this makes mutation from individuals function to adapt
• Technique of fingerprint originally for southblots that include DNA that has been digestion, transferred, hybridization and detected through patterns where the band varies if the 2 are monozygotic