DNA and RNA: Structure and Function

• Nucleic acids are long polymers of nucleotides and DNA stores genetic information.

Nucleic Acids: DNA vs RNA

• DNA acts as the genetic material in most organisms
• RNA mostly helps in the transfer and expression of information.
• DNA is chemically and structurally more stable, making it a better genetic material
• RNA was the first to evolve, and DNA was derived from it
• RNA functions as a messenger, adapter, structural, and sometimes catalytic molecule.

DNA Structure

• The double-stranded helix is held by hydrogen bonds between bases on opposite strands.
• Adenine (A) pairs with Thymine (T) via two hydrogen bonds
• Guanine (G) pairs with Cytosine (C) via three hydrogen bonds.
• Base pairing confers a unique property, making the strands complementary for predictable sequencing.
• DNA is a long polymer of deoxyribonucleotides
• DNA length is defined by the number of nucleotides or base pairs.
• Bacteriophage φX174 has 5386 nucleotides.
• Bacteriophage lambda has 48502 base pairs.
• E. coli has 4.6 x 10^6 base pairs.
• Human DNA has 3.3 x 10^9 base pairs.

Polynucleotide Chain Structure

• A nucleotide has three components: nitrogenous base, pentose sugar, and a phosphate group.
• Nitrogenous bases are either Purines (Adenine and Guanine) or Pyrimidines (Cytosine, Uracil and Thymine).
• Cytosine is common to both DNA and RNA, while Thymine is exclusive to DNA and Uracil to RNA.
• A nitrogenous base links to the 1'C of a pentose sugar through an N-glycosidic link, forming a nucleoside.
• A phosphate group links to the 5'C OH of a nucleoside via a phosphoester linkage, forming a nucleotide.
• Nucleotides link through a 3'-5' phosphodiester linkage, forming a dinucleotide, and further, a polynucleotide chain.
• A polynucleotide chain has a 5' phosphate end and a 3' hydroxyl end.
• The sugar and phosphates form the backbone of the chain with nitrogenous bases projecting.
• RNA has an additional -OH group at the 2' position of the ribose and uses uracil instead of thymine.

DNA Double Helix Features

• Composed of two polynucleotide chains with a sugar-phosphate backbone
• Bases project inward.
• The two chains run anti-parallel (5' to 3' and 3' to 5').
• Bases pair via hydrogen bonds (A=T, G≡C), a purine always pairs with a pyrimidine to maintain a consistent distance
• The chains are coiled in a right-handed fashion.
• A helix pitch is 3.4 nm with about 10 base pairs per turn, with approximately 0.34 nm between a base pair in a helix
• The base pairs stack over one another thereby conferring stability to the helical structure.
• Friedrich Meischer identified DNA as an acidic substance in the nucleus in 1869 and named it 'Nuclein’.

DNA Packaging

• Distance between two base pairs is 0.34 x 10^-9 m.
• The length of DNA double helix in a mammalian cell is approximately 2.2 meters
• DNA (negatively charged) is held with proteins (positively charged) in the nucleoid.
• In eukaryotes, positively charged basic proteins called histones organize DNA.
• Histones are rich in lysine and arginine.
• Eight histones form a histone octamer.
• Negatively charged DNA wraps around the histone octamer forming a nucleosome.
• A nucleosome contains 200 bp of DNA helix.
• Nucleosomes repeat to form chromatin, which appears as 'beads-on-string'.
• Chromatin is packaged to form chromatin fibers, which coil and condense into chromosomes during metaphase.
• Additional proteins are required for higher-level packaging of chromatin and are called Non-histone Chromosomal (NHC) proteins.
• Euchromatin stains lightly and is loosely packed
• Heterochromatin stains darkly and is densely packed, and transcriptionally inactive.

The Search for Genetic Material

• DNA acts as a genetic material was proven by 1926.
• Gregor Mendel, Walter Sutton, and Thomas Hunt Morgan narrowed the search to chromosomes.

Transforming Principle

• Frederick Griffith's 1928 experiments with Streptococcus pneumoniae:
• S strain (smooth, virulent) kills mice
• R strain (rough, non-virulent) does not.
• Heat-killed S strain does not kill mice.
• Heat-killed S strain mixed with live R strain kills mice
• Live S strain bacteria were recovered from dead mice.
• Griffith concluded that R strain transformed into S strain
• A transforming principle from the heat-killed S strain enabled R strain to synthesize a polysaccharide coat and become virulent.

Biochemical Characterization of Transforming Principle

• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1933-44) determined the transforming principle.
• DNA alone from the S bacteria caused R bacteria to transform.
• Protein-digesting enzymes (proteases) and RNA-digesting enzymes (RNases) did not affect transformation
• Digestion with DNase inhibited transformation, suggesting DNA caused the transformation.
• Therefore, DNA is the hereditary material.

The Genetic Material is DNA

• Alfred Hershey and Martha Chase (1952) proved DNA is the genetic material.
• Worked with bacteriophages (viruses infecting bacteria).
• Bacteriophage attaches to the bacteria and its genetic material enters the bacterial cell.
• The viral genetic material is treated as its own by the bacteria and manufactures virus particles.
• Some viruses were grown on medium containing radioactive phosphorus (32P) and some on radioactive sulfur (35S).
• Viruses grown with radioactive phosphorus contained radioactive DNA but not radioactive protein.
• Viruses with radioactive sulfur contained radioactive protein but not radioactive DNA.
• Radioactive phages infected E. coli bacteria.
• Viral coats were removed by agitation, and virus particles were separated via centrifuge.
• Bacteria infected with radioactive DNA viruses were radioactive.
• Proteins did not enter the bacteria from the viruses.
• DNA is the genetic material transferred to bacteria from viruses.

Properties of Genetic Material

• RNA is the genetic material in some viruses (e.g., Tobacco Mosaic virus) however DNA is predominant.
• A molecule that acts as genetic material must:
• Generate its replica (Replication).
• Be stable chemically and structurally.
• Provide scope for slow changes (mutation) for evolution.
• Express itself in the form of 'Mendelian Characters'.
• DNA and RNA both have the ability to direct duplications due to base pairing and complementarity rule.
• The genetic material should be stable enough not to change with life cycle stages, or physiology.
• Transforming principle was evident in Griffith’s experiment.
• Two DNA strands come together when heated if appropriate conditions are provided.
• RNA has a reactive 2'-OH group at every nucleotide, making it labile and easily degradable, and therefore is more reactive.
• The presence of thymine instead of uracil in DNA confers additional stability.
• RNA mutates faster since it is unstable.
• RNA can directly code for the synthesis of proteins and express characters easily.
• DNA depends on RNA for protein synthesis
• Both RNA and DNA can function as genetic material DNA is preferred for storing genetic information due to its stability, while RNA is ideal for genetic transmission due to its dynamic nature.

RNA World

• RNA was the first genetic material.
• RNA acted as a genetic material and a catalyst for reactions such as metabolism.
• RNA evolved into stable DNA with chemical modifications.
• DNA being double stranded resists changes by repair

Replication

• Watson and Crick proposed that the two DNA strands would separate, acting as templates for new complementary strands i.e. semiconservative DNA replication.
• Evidence for semiconservative replication was shown first in E. coli
• Matthew Meselson and Franklin Stahl performed the following experiment in 1958:
  • E. coli was grown in a medium containing 15NH4Cl (15N is the heavy nitrogen isotope) as the nitrogen source.
  • Over generations, 15N was incorporated into newly synthesized DNA.
  • Heavy (15N) DNA distinguished from normal DNA by centrifugation in a cesium chloride (CsCl) density gradient.
  • 15N is not a radioactive isotope.
• Cells were transferred to a medium with normal 14NH4Cl and samples extracted over time.
• DNA samples were separated on CsCl gradients.
• Recall centrifugal force, and why molecules with higher mass/density sediment faster.
• Conclusion: DNA is extracted one generation after transfer from 15N to 14N, that is, after 20 mins, and had a hybrid i.e. intermediate density. extracted at another generation had equal amounts of the hybrid DNA and the light “N,
• Taylor and colleagues proved DNA in chromosomes replicates semiconservatively.

The Machinery and the Enzymes

• DNA-dependent DNA polymerase uses a DNA template to catalyze deoxynucleotide polymerization. -E. coli completes replication in 18 minutes resulting in 2000 bp per second, and maintains a high degree of accuracy.
• Deoxyribonucleoside triphosphates have a dual purpose:
• As substrates for polymerisation
• Provide energy for the polymerisation reaction.
• Two DNA strands cannot be fully separated, so replication occurs in a replication fork.
• DNA polymerases catalyze polymerization in one direction (5' to 3').
• One strand (3' to 5' polarity) undergoes continuous replication
• The other (5' to 3' polarity) undergoes discontinuous replication, forming fragments joined by DNA ligase.
• DNA polymerases cannot initiate replication randomly - a definite region or origin of replication is required in E. coli DNA
• Vectors are required for DNA propagation, providing the origin of replication.
• In eukaryotes, DNA replicates during the S-phase of the cell cycle
• Failure causes polyploidy.

Transcription

• The genetic information is copied from one strand of DNA into RNA and it is called transcription.
• Complementarity governs transcription, where uracil substitutes for thymine when forming a base pair.
• Only a DNA segment and one of the strands are copied into RNA.
• If both strands were copied, different sequences for two different proteins would be coded, creating complexity.
• Additionally, two RNA molecules would form a double-stranded RNA preventing translation.

Transcription Unit

• A transcription unit in DNA is defined by three regions
• A promoter
• The structural gene
• A terminator
• There is a convention in defining the two strands of DNA in the structural gene of a transcription unit.
• Since the two strands have opposite polarity, the strand with polarity 3’ to 5’ is the template strand
• The other strand (5’ to 3’) with sequence same as RNA is the coding strand
• The promoter and terminator flank the structural gene in a transcription unit. -Promoter is located towards 5’ and provides binding site for RNA polymerase. -Terminator is located towards 3’ and defines the end of transcription.

Transcription Unit and The Gene

• Genes are located on the DNA.
• A cistron is a DNA segment coding for a polypeptide
• The structural gene can be monocistronic (eukaryotes) or polycistronic (prokaryotes).
• In eukaryotes, genes are interrupted. -Coding sequences are exons and intervening sequences are introns. -The split-gene arrangement complicates gene definition and inheritance of character is affected by promoter and regulatory sequences of a structural gene.

Types of RNA and Process of Transcription

• In bacteria, mRNA, tRNA, and rRNA are used for protein synthesis with mRNA providing the template.
• tRNA brings amino acids and reads the genetic code, while rRNAs play structural and catalytic roles
• RNA polymerase binds to promoter and initiates transcription using nucleoside triphosphates, i.e. initiation.
• It polymerizes through complementarily thereby continuing elongation.
• Enzyme moves to terminator, the nascent RNA falls off resulting in termination.
• RNA polymerase associates with initiation-factor (σ) and termination-factor (ρ) to initiate/terminate the transcription.
• Bacteria mRNA does not require processing, for translation i.e. transcription and translation are coupled.

Eukaryotic Transcription

• Eukaryotes have three RNA polymerases:
• RNA polymerase I transcribes rRNAs (28S, 18S, and 5.8S).
• RNA polymerase III transcribes tRNA, 5srRNA, and snRNAs (small nuclear RNAs).
• RNA polymerase II transcribes precursor of mRNA (heterogeneous nuclear RNA, or hnRNA).
• Primary transcripts contain exons and introns.
• Splicing removes introns and joins exons in order.
• hnRNA undergoes capping (addition of methyl guanosine triphosphate to the 5' end) and tailing (addition of adenylate residues at the 3' end).
• Fully processed hnRNA, now mRNA, transports out of the nucleus for translation.

Genetic Code

• During replication and transcription a nucleic acid was copied to form another nucleic acid.
• Translation involves the transfer of genetic information from a polymer of nucleotides to polymerize amino acids
• Changes in nucleic acids (genetic material) responsible for change in amino acids
• There is a genetic code directs the sequence of amino acids during synthesis of proteins.
• If determining biochemical nature of genetic material and structure of DNA was exciting, the proposition and deciphering of genetic code were most challenging in a very true sense it required involvement of scientists from several disciplines - physicists, organic chemists, biochemists and geneticists.
• George Gamow argued 4 bases code for 20 amino acids and code consists of a combination of three nucleotides with permutation generating 64 codons thereby leading to many more codons than required.
• Instruments synthesise RNA molecules with defined combinations.
• Marshall Nirenberg's cell-free system for protein synthesis helped to decipher the code.
• Severo Ochoa enzyme (polynucleotide phosphorylase) uses enzymatic synthesis of RNA with defined sequences.
• There are 61 codons for amino acids and 3 stop codons
• Some amino acids are coded by more than one codon thereby is degenerate.
• The codon is read in mRNA in a contiguous fashion without punctuations.
• The code is nearly universal with some exceptions found.
• AUG has dual functions with codes for Methionine (met) and as an initiator codon.
• UAA, UAG, UGA are stop terminator codons.

Mutations and Genetic Code

• The relationships between genes and DNA are understood by mutation studies.
• Large deletions and rearrangements in DNA segment result in a gene and function loss or gain
• If there is a change of single base pair in gene for beta globin chain it results in change of the amino acid residue glutamate to valine which is known as the point mutation, in diseased condition referred as sickle cell anemia.
• Insertion or deletion changes lead to mutations

tRNA- the Adapter Molecule

• Francis Crick proposed a mechanism reads the code and links it amino acids to read it uniquely.
• tRNA bears an anticodon loop and amino acid acceptor end thereby is specific for each amino acid with an initiator tRNA for initiation with the secondary structure looking like clover leaf
• During insertion or deletion of three or its multiples bases lead to insertion or deletion in single or multple codon
• There no changes in the amino acids, and reading frame remains unaltered.

Translation

• Refers to the process of polymerisation of amino acids to form a polypeptide.
• The order and sequence of amino acids are defined by the sequence of bases in the mRNA.
• A peptide bond requires energy and amino acids are activated in the presence of ATP to form tRNA.
• If the two tRNAs are close formation of a peptide bond occurs requiring catalysts that is the ribosome.
• Ribosome subunits consist of structural RNAs and about 80 different proteins in two subunits
• If small subunits encounters mRNA the translation to protein is initiated.
• There are 2 sites on large subunit for subsequent amino acids to and and is close enough to form a peptide bonds

Regulation of Gene Expression

• Occurs at various levels for polypeptide formation.
• Eukaryotes regulation exerted at:
  • transcriptional level (formation of primary transcript).
  • processing level (regulation of splicing).
  • transport of mRNA.
  • translational level.
• Operon model has genes in a cell expressed for a particular function with beta-galactosidase synthesized by E. coli catalyze lactose hydrolysis for energy
• Genes are regulated for conditions due to conditions.
• In prokaryotes, transcriptional initiation controls gene expression.
• Accessory proteins affect RNA polymerase
• Regulatory proteins act as activators and repressors.
• Prokaryotic DNA accessibility regulated by interaction of proteins with operators.
• Each operon has a specific operator and repressor with the lac operator interacting with lac repressor.

The Lac Operon

• Elucidation of the lac operon was a result of a close association and this Jacob-Monod system is transcriptionally regulated
• Lac operon has polycistronic structural gene regulated by common promoter and regulatory genes.
• Lac operon has regulatory gene (i gene) with structural genes termed(z, y, and a) and that are the repressor codes, beta-galactosidase (β-gal) gene codes and the permease gene codes
• All three gene products in lac operon required for lactose metabolism.
• Lactose induces switching and off of operon is the lac operon
• RNA polymerase is transcribed when beta galactosidase lactose occurs, and the repressor prevents RNA polymerase transcripts.
• The lac Operon is a form of negative regulation with the operon a control of positive regulation.

Human Genome Project

• DNA determines the genetic information of organisms in sequences.
• Genetic make-up lies in the DNA and assumptions led to quest of DNA sequence of human genome.
• Human Genome Project (HGP) launched in 1990 as “mega project”.
• Determine 3 x 109 bp, with its cost per bp at US $ 3 with approximately 9 billion US dollars.
• Goals of HGP: -Identify all 20,000-25,000 genes in human DNA. -Determine the sequences of 3 billion chemical base pairs that make up human DNA. -Store this information in databases, and improve tools for data analysis. -Transfer related technologies to other sectors -Address the ethical, legal, and social issues (ELSI) from the project.
• It was a 13 year project that was coordinated from the Department of Energy and Nation Institute of Health with wellcome trust (UK) and contributions came.
• Human Genome Project: Disorders for humans lead to revolutionary treatment methodolgies include genes expressed, by isolating and convert total DNA in random order etc, these were sequenced using DNA sequencers that that has a framework to developing amino acids
• The end result is that These all give some overlapping regions that are used for alignments
• The sequences are that were assigned to each chromosome as a result there has been another challenge to that assigning to the genetic and physical maps of the genome
• This was generated using for polymorphisum information by some repetitive DNA sequences ( microsatellites)

Salient Features of Human Genome

• Human genome project are as follows: genome conatains 3.164.7 millon
• Consist of 3000 bases with that are vary greatly with genes being dystrophin at 2.4 million bases. the estimated number in around to approximately lower that estiminations the nuceoltides are same in all the people
• Many genes have unknown functions, less than 2 percent codes for proteins with a repariative that are a large human genomnes -they there are stretchers that are in a direct codigf that helps shed the chromosoms and have dynmiams

Applications and Future Challenges

derive research from the our understasdfing that tasks need from one and expertises and is related to biology in past genes at time

• With whole genome sequences and high tech we can apporach ystematically on much broader
• Study the genes study transcirpts tissue and tumor or thousnd that protien will interconnect chemistry in

Applications and Future Challenges

derive research from our understandiing that tasks need expertises and biology in past genes at time

• With whole genome sequences and high tech we can apporach ystematically on much broader Study the genes transpirt tissue and tumor or protien will interconnect cheistry

DNA Fingerprinting

• As stated in the preceding section, 99.9 per cent of base sequence among humans is the same
• Assuming human genome as 3 × 10º bp
• There are sequences of DNA which make every individual unique in their phenotypic appearance
• DNA fingerprinting is a quick way to compare the DNA sequences of any two individuals
• DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA
• Since DNA from every tissue (such as blood, hair-follicle, skin, bone, saliva, sperm etc.), from an individual show the same degree of polymorphism, they become very useful identification tool and for the case of forensic

DNA

• It is essential that we understand what DNA polymorphism means in simple terms, Polymorphism (variation at genetic level) arises due to mutations New mutations may arise in an individual either in somatic cells or in the germ cells (cells that generate gametes in sexually reproducing organisms) Again recall the definition of alleles from Chapter 4) sequence variation has traditionally been described as a DNA polymorphism if more than one variant in simple terms population is referred to as DNA polymorphism
• There is a variety of different types of polymorphisms ranging from single nucleotide change to very large scale changes
• Technique by Alec fingerprints a Satellite technique