Molecular Basis of Inheritance: DNA Structure

Molecular Basis of Inheritance

• Chapter explores patterns of inheritance and their genetic basis.
• DNA is confirmed genetic material for most organisms.
• Nucleic acids are polymers of nucleotides.
• Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are two types of nucleic acids.
• DNA serves as the genetic material in most organisms.
• RNA acts as a messenger, adapter, structural, and catalytic molecule, and as genetic material in some viruses.
• Chapter covers structure of DNA, replication, transcription, genetic code, protein synthesis, and regulation.
• Complete nucleotide sequence of human genome during last decade has set in a new era of genomics.

The DNA

• DNA is a long polymer of deoxyribonucleotides; length is defined by number of nucleotides or base pairs.
  • Bacteriophage φX174 has 5386 nucleotides.
  • Bacteriophage lambda has 48502 base pairs.
  • Escherichia coli has 4.6 x 10^6 base pairs.
  • Human DNA has 3.3 x 10^9 base pairs.

Structure of Polynucleotide Chain

• Nucleotide's components: nitrogenous base, pentose sugar, and phosphate group.
• Nitrogenous bases come in two types, Purines (Adenine, Guanine) and Pyrimidines (Cytosine, Uracil, Thymine).
• Cytosine is in both DNA/RNA, Thymine in only DNA, and Uracil replaces Thymine in RNA.
• A nitrogenous base links to 1'C of pentose sugar via N-glycosidic linkage, forming a nucleoside.
  • Examples: adenosine, deoxyadenosine, guanosine, deoxyguanosine, cytidine, deoxycytidine, uridine, deoxythymidine
• Phosphate group links to 5'C OH of a nucleoside through phosphoester linkage.
• Two nucleotides link through 3'-5' phosphodiester linkage.
• Polymer has a free phosphate at the 5' end of the sugar (5' end) and a free OH at the 3'C sugar (3' end).
• Backbone: sugar and phosphates, with nitrogenous bases projecting from it.
• Each nucleotide residue in RNA has an additional -OH group at the 2' position
• Uracil found in RNA instead of thymine (5-methyl uracil).
• DNA was first identified by Friedrich Meischer in 1869 as an acidic substance, named 'Nuclein'.
• James Watson and Francis Crick proposed the Double Helix model in 1953, from X-ray diffraction data by Maurice Wilkins and Rosalind Franklin.
• Key feature was base pairing between two polynucleotide chains, based on Erwin Chargaff's observation that ratios of Adenine to Thymine and Guanine to Cytosine are constant.
• Base pairing makes the strands complementary; one strand's sequence predicts the other.
  • A parental DNA strand serves as a template to produce identical daughter DNA.

Salient Features of Double-helix Structure of DNA

• Two polynucleotide chains form the helix, with sugar-phosphate backbone and inward-projecting bases.
• Two chains run antiparallelly: one 5'→3', the other 3'→5'.
• Bases pair via hydrogen bonds: Adenine with Thymine (two bonds), Guanine with Cytosine (three bonds); a purine always pairs with a pyrimidine.
• Two chains coil in right-handed fashion; helix pitch is 3.4 nm with roughly 10 bp per turn.
• Distance between a bp is 0.34 nm.
• One base pair plane stacks over the other in the double helix, plus H-bonds provide stability to helix structure
• DNA double helix simplicity explained genetic implications and Francis Crick proposed the Central dogma where genetic information flows from DNA to RNA to Protein

Packaging of DNA Helix

• Distance between two consecutive base pairs is 0.34 x 10^-9 m, if the length of DNA double helix in a typical mammalian cell is calculated at 2.2 meters.
• Prokaryotes lack defined nucleus; DNA (negatively charged) held with positive proteins in 'nucleoid' and organized in large loops.
• Eukaryotes are structured more complex with basic proteins called histones, which are positively charged due to lysine and arginine and combine to form a histone octamer.
• Negatively charged DNA wraps around histone octamer to form nucleosome: a typical nucleosome contains 200 bp of DNA helix.
• Nucleosomes repeat as chromatin in the nucleus.
• Chromatin observed as 'beads-on-string' under an electron microscope.
• Beads-on-string structure are packaged into chromatin fibers, which are further coiled and condensed at metaphase stage of cell division to form chromosomes.
• Non-histone Chromosomal (NHC) proteins form the proteins which require higher level packaging of chromatin.
• Euchromatin: loosely packed chromatin regions (stain light); Heterochromatin: densely packed chromatin regions (stain dark).
• Euchromatin is transcriptionally active; heterochromatin is inactive.

The Search for Genetic Material

• Nuclein discovery and inheritance waited long to prove DNA as genetic material
• By 1926, genetic inheritance had reached the molecular level, previous discoveries narrowed the search to the chromosomes located in the nucleus of most cells however question of genetic material was not answered

Transforming Principle

• Frederick Griffith in 1928 showed miraculous transformation in Streptococcus pneumoniae bacteria turning living to a different physical form
• S strain produced smooth shiny colonies and R strain produced rough colonies, mice infected with S died of pneumonia and R did not cause pneumonia
  • injected S strain into mice made mice die
  • injected R strain into mice made mice live
• Griffith could kill the bacteria by heating them; heat killed the bacteria however when injected with harmless R it made the mice die, recovering the living S strain from the dead mice
• R strain was transformed by the heat-killed S strain allowing R strain to synthesize a smooth polysaccharide coat making it virulent and transferring genetic material without defining the biochemical nature

Biochemical Characterisation of Transforming Principle

• Prior to work of Oswald Avery, Colin MacLeod and Maclyn McCarty (1933-44), genetic material was considered a protein; they wanted to determine the biochemical nature of Griffiths transforming principle by purifying from heat killed S cells
• Discovered DNA isolated from S bacteria made R transform
• Also discovered protein digesting enzymes or RNA digesting enzymes from S cells did not stop transformation, digestion with DNase did, determining that DNA is the hereditary material but was still not convinced

The Genetic Material is DNA

• Unequivocal proof was found by Alfred Hershey and Martha Chase (1952) that viruses infect bacteria called bacteriophages by attaching and inserting genetic material tricking bacteria into manufacturing virus particles
• Viruses were grown with radioactive phosphorus/sulfur, radioactive phosphorus had radioactive DNA and sulfur had protein
• Then radioactive phages allowed E Coli infection, viral coats removed with a blender and separated via centrifuge and found radioactive DNA infected bacteria to become radioactive, radioactive proteins were not indicating proteins did not enter bacteria meaning DNA is the genetic material

Properties of Genetic Material (DNA versus RNA)

• Viruses have genetic RNA and predominant genetic DNA
• A molecule acting as genetic material must fulfill
  • replicate itself
  • be chemically and structurally stable
  • provide scope for slow mutations for evolution
  • express itself in the form of Mendelian Characters
• Both DNA and RNA have the ability to direct duplicates due to base pairing and complementation
• Genetic material must be stable and not change with organisms cycle, age or physiology
• Griffith's 'transforming principle' was heat stable preserving its genetic properties
• DNA being complementary can come back together even if separated by heat
• RNA has reactive 2’ -OH which is easily degradable and known to be catalytic therefore unstable structurally and chemically compared to DNA
• Fact that it has Thymine which confers more stability to DNA, requiring detailed understanding of a repair process in higher classes
• Both can mutate, RNA mutates faster due to instability allowing for fast evolution, short life and expression of characters through protein as DNA is dependent on synthesis of proteins
• Both function as more stable genetic material storing information as RNA transmits it

RNA World

• RNA was the first genetic material supported by essential life processes like metabolism, translation, splicing
• Catalytic RNA catalysts exist and being one implies instability, becoming a more stable DNA after chemical modifications
• DNA is double stranded with complementary strands which resists change, requiring process of repair

Replication

• Watson and Crick Proposed scheme of DNA replication after double helix structure
• Two stands separate and act as templates forming new complementary structure of template
• Is called semiconservative DNA replication where each one has one parental and one new strand

The Experimental Proof

• DNA replicates semiconservatively shown in Escherichia coli, later in higher organisms, plants, and human cells by Matthew Meselson and Franklin Stahl in 1958
  • Grew E. coli with heavy isotope of nitrogen making nitrogen only source for generations, incorporating into newly synthesised DNA
  • Molecule was distinguished from normal via cesium chloride (CsCl) based
  • Cells with normal 14NH4Cl multiplied and had samples extracted as double stranded helices and were separated via CsCl gradients, showed high density in normal generation
• Transferred one culture from 15N to 14N medium with 20 minutes extraction containing E Coli divided and hybridized density and after another 40 it measured equal amounts
• Radioactive thymidine used to detect distribution of newly synthesized DNA on Vicia faba beans in chromosomes by Taylor and colleagues in 1958 proved that the DNA also replicates semiconservatively

The Machinery and the Enzymes

• Living organisms use catalyst sets referring the main enzyme as DNA-dependent DNA polymerase using DNA template to catalyze polymerization of deoxynucleotides making enzymes efficient as catalyzing large amounts in short time
• E Coli has only 4.6 x 10^6 bp comparing human whose content has 6 x 10^9 bp, completes process in 18 minutes averaging per second is 2000 bp along with reactions needing high accuracy mutations and are energetically expensive
• Deoxyribonucleoside triphosphates are dual serving as an additional substrate providing energy for polymerase reactions for terminal phosphates which are high energy same as ATP
• Since DNA strands can not separate the process of copying genetic information occurs within only a small DNA helix opening defined as replicating fork
• DNA dependent DNA polymerase has only direction of catalyzing defined as 5’->3’
• Leads to replication of continuous strands, template with 3’-> 5 and discontinuous with polarity of 5’->3’ and discontinuously formed requires DNA ligase
• DNA polymerization can not initially start without process and doesnt randomly happen, definite region in E Coli DNA originates defines as origin requires vector if piece needs to propagate recombinant DNA

Transcription

• Copying one strand of genetic information from DNA into RNA is transcription; complimentarily governs the process and transcription with adenosine now creating base pairing with uracil instead of thymine
• Unlike replication which sets total duplicated DNA, only a transcription DNA segment copies into RNA. A single segment and strand is copied therefore necessitates defining region boundaries and DNA strand definition with the boundary
• Transcription copies only one strand because
  • if both templates both RNA molecules with different sequences would produce proteins with different acids meaning one DNA segment meaning coding
  • and complimentary molecules would be double stranded preventing translation and becoming futile

Transcription Unit

• Transcription defined by the DNA regions
  • promoter
  • structural gene
  • terminator
• Two DNA strands in the structural gene will make polymerization 5’->3’, 3’->5 can act as a template referring to the other with the sequence being same but displaced during transcription as coding
• Reference is made with the coding strand for transcription
• Promoter and terminator are flanks in the structural gene locating promoters towards 5’ end and structural upstream defining template and coding strands, also by terminators is coding being located 3 end also defining the processes
• The expression and gene is functional but difficult to locate due
• Definition with RNA rRNA makes defining a challenge which is polypeptide segment as cistron
• Transcript is typically said as monocistronic in and also mostly in eukaryotes
• Monocistronic structural genes contain interrupt coding so they are split
• Exons are coding defined in mature sequences
• Introns do not appear as RNA

Types of RNA and process of Transcription

• Bacteria has three main types of RNA, mRNA (messenger). tRNA (transfer); and rRNA (ribosomal) needing to synthesize proteins which template which bring acid, read to genetically play structural and catalytic role during translation
• Single DNA-dependent RNA polymerses can catayze all forms transcription where polymerase initiates with nucleoside triphosphytes as single factors
• Template can follow compliment rule for elongation however short stretch has to remain bound as RNA will result termination of the nascent

Genetic Code

• During replication and transcription you replicate a nucleic into another where process is easier to conceptualize but translating into a protein is not nor does there exist data supporting this
• Though data exists that alterations in genetic code has an impact which created suggestion can proteins
• Bio chemistry was exciting with challenges
• Scientist George Gamow argued there are four base codes so combine therefore there is also code amino made of bold suggesting

Mutations and genetic code

• Understand where small can alter, delete base which understood considering
• Statement is made genetic and
• Statement the
• Similarly
• If we statements
• Repeated
• The change from
• However the to is such alter
• Molecules clear
• Read coded the
• Amino structural is
• Are from one in one in

Translation

• Polymeration of amino acids to form polypeptide defines process defined by the sequence of bases in the mRNA
• Amino acids joined with a peptide bond requires forming amino charged is specific brought forming