Molecular Basis of Inheritance - Important Points, Summary, Revision, Highlights

#DNA #Polynucleotide Chain #Double Helix Model #Packaging of DNA #Replication #Transcription #Genetic Code #Mutation #Translation #Central Dogma #Regulation #Lac Operon #HGP #DNA Fingerprinting

The Deoxyribonucleic Acid (DNA)

Except for some viruses, such as Tobacco mosaic virus (TMV), DNA is the genetic material found in the majority of organisms.

RNA mostly acts as a messenger, an adapter, and has a catalytic function.

The length of DNA is determined by the number of base pairs (bp) or nucleotides.

Human DNA (haploid): 3.3 x 10⁹ bp

Bacteriophage 𝜙 X 174 has 5386 nucleotides.

Bacteriophage 𝝀: 48502 bp

E. coli – 4.6 x 10⁶ bp

Friedrich Meischer identified DNA present in the nucleus in 1869 and named it ‘Nuclein’.

Frederick Griffith in 1928 demonstrated that a “transforming principle” could be transferred from heat-killed S-strain of Streptococcus pneumoniae to R-strain, allowing it to produce a smooth polysaccharide coat and become virulent in infected mice.

Oswald Avery, Colin MacLeod, and Maclyn McCarty determined that only DNA is the biochemical nature of the “transforming principle” responsible for transformation.

Hershey and Chase in 1952 proved that DNA is the genetic material by infecting the bacteria E. coli with bacteriophages grown in radioactive phosphorus (32P) and radioactive sulfur (35S). The phosphorus labeled the DNA of the bacteriophage, which was transferred to the bacterial cells, while the sulfur labeled the protein coat of the bacteriophage. As a result, radioactivity was not detected in the bacterial cell.

Structure of a Polynucleotide Chain

Each nucleotide is composed of three components:

1. Nitrogenous Bases:

2. Purines - Adenine (A) and Guanine (G) are present in both DNA and RNA.

3. Pyrimidines- Cytosine (C) and Thymine (T) in DNA and Cytosine and Uracil in RNA. Thymine is also known as 5-methyl uracil and it is accounted for more stability of DNA molecule.

4. Sugar:

• Pentose sugar:
  • Ribose in RNA (ribonucleic acid)
  • Deoxyribose in DNA

3. Phosphate Group

Nucleoside: a nitrogenous base linked to the hydroxyl group of the 1’ C of the pentose sugar via an N-glycosidic bond.

Nucleotide: A phospho-ester bond attaches the phosphate group to the hydroxyl group present at the 5’ C of the nucleoside.

Structure of a Nucleotide

A 3’-5’ phosphodiester bond links two nucleotides together to form a dinucleotide, and the chain continues to grow, forming a polynucleotide.

A Nucleotide Chain

Double Helix Model of the Structure of DNA

Watson and Crick proposed the double helix structure of DNA in 1953.

Ervin Chargaff observed that the ratio of Adenine to Thymine and Guanine to Cytosine is one and remains constant.

Two polynucleotide chains make up DNA, with a sugar-phosphate backbone and bases towards the inside.

One chain has a 5’→3’ polarity and the other has a 3’→5’ polarity.

Base pairs (bp) are formed by hydrogen bonding between nitrogenous bases of both the polypeptide strands.

Always a purine base of one nucleotide chain is linked to a pyrimidine base of another nucleotide chain, or vice versa, to form a base pair.

Adenine pairs with Thymine (or Uracil in RNA) by two hydrogen bonds (A=T)

Guanine pairs with Cytosine by three hydrogen bonds (G-C-3H)

The two polypeptide chains are coiled in a right-handed direction.

10 base pairs have a distance of 0.34 nm, and the pitch of the helix is 3.4 nm.

The stability of the helical structure is conferred by the stacking of base pairs one above the other.

DNA Double Helix Structure

Packaging of DNA Helix

In prokaryotes, DNA is organized as a large loop in the nucleoid region. Positively charged proteins bind together the negatively charged DNA.

In eukaryotes, DNA is organized into complex structures called chromosomes. The DNA is wrapped around the core of an octamer, composed of 8 histone molecules, to form a nucleosome.

Histones are positively charged proteins, as they are abundant in basic amino acids such as lysine and arginine.

There are 5 types of histone proteins:

• H1
• H2A
• H2B
• H3
• H4

The Histone octamer consists of two molecules of four histone proteins and is essential for gene regulation.

A nucleosome is a repeating unit in chromatin that prevents DNA from becoming tangled.

The Nucleosome contains approximately 200 base pairs of DNA.

Non-histone chromosomal proteins (NHC) help to facilitate additional structuring of chromatin.

Euchromatin: These are transcriptionally active areas where chromatin is loosely packed and they take up a light stain.

Heterochromatin: These are transcriptionally inactive areas of chromatin that are densely packed and appear dark when stained.

Replication

Watson and Crick suggested that the replication of DNA is semiconservative.

Meselson and Stahl proved experimentally in 1958 that DNA replicates semi conservatively.

Taylor et al proved in another experiment on Vicia faba (fava beans) using radioactive thymidine that DNA replication is semiconservative.

DNA polymerase catalyses DNA replication and can only polymerise in the 5’→3’ direction.

Replication is initiated at the origin of replication.

Deoxyribonucleoside triphosphate (dNTP) provides energy for the polymerisation reaction and serves as a substrate.

A replication fork is formed when a small part of DNA opens up, allowing for replication to occur.

The template strand for the leading strand has a 3’→5’ polarity and is referred to as the leading strand template, while the strand undergoing continuous replication has a 5’→3’ direction and is referred to as the leading strand.

The template strand of the lagging strand has 5’→3’ polarity, and replication is discontinuous in the other strand.

The Okazaki fragments, which are discontinuous, are joined together by the enzyme DNA ligase.

In eukaryotic cells, the replication of DNA occurs during the S-phase of the cell cycle.

If cell division does not occur following replication, it can lead to polyploidy of chromosomes.

Transcription

In the process of transcription, the genetic information present in the DNA is transcribed to RNA.

Only one segment of DNA is transcribed into RNA

In RNA, Uracil is present instead of Thymine which is present in DNA

Transcription of DNA includes three regions: a promoter, the structural gene, and a terminator.

RNA polymerase catalyses the transcription, and the direction of transcription is the same as that of replication by DNA polymerase, i.e. 5’→3’ direction.

Antisense Strand: It has 5’→3’ polarity, which acts as a template for DNA formation, also called template strand.

Coding strand: it has 5’→3’ polarity, which has the sequence similar to the newly formed RNA, except that Thymine is replaced by Uracil in RNA, also referred to as the sense strand.

Promoter: It is situated at the 5’ side of the structural gene or upstream (with respect to the coding strand). Here, RNA polymerase binds to start the transcription.

• Structural gene: The region between a promoter and a terminator. A cistron is a segment of DNA that codes for a single polypeptide. The structural gene is monocistronic in eukaryotic cells and polycistronic in prokaryotic cells.

Terminator: It is located at the 3’ end of the coding strand and marks the end of the transcription process.

Split genes are eukaryotic structural genes with interrupted coding sequences, also known as monocistronic genes.

Exons: Coding sequences that are present in both mature and processed RNA.

Introns: intervening sequences that are not present in the mature and processed RNA.

Transcription in a Prokaryotic Cell

In bacteria, a single DNA-dependent RNA polymerase is responsible for the transcription of mRNA, tRNA, and rRNA.

There are three steps of transcription:

1. Initiation: RNA polymerase binds to initiation factor, 𝞂 (sigma) at the promoter site to start the process of transcription

2. Elongation: RNA Polymerase is only capable of extending the length of the RNA strand.

3. Termination: When the RNA polymerase reaches the terminator region, it binds with the Terminator Factor, Rho (𝜌) to terminate the process, resulting in the nascent RNA falling off.

Process of Transcription in Bacteria

In bacteria, mRNA does not need additional processing and since the nucleus and cytosol are not separate, translation is combined with transcription and begins before the entire transcription of mRNA is finished.

Transcription in a Eukaryotic Cell

There are three RNA polymerases (I, II, and III) that catalyze the transcription of different RNAs in a eukaryotic cell.

1. RNA Polymerase I:

• Transcription of rRNA (28S, 18S, 5.8S)

2. RNA polymerase II- transcription of pre-mRNA (precursor of mRNA), which is a form of hnRNA

3. RNA Polymerase III: Transcription of tRNA, 5S rRNA, and snRNA (Small Nuclear RNA)

The primary transcript is non-functional and has exons interrupted by introns.

Splicing: The process of removing introns and joining together exons in a defined sequence.

Capping and Tailing - Additional processing of hnRNA. In Capping, a Methyl Guanosine Triphosphate is added to the 5’ end of hnRNA. In Tailing, 200-300 Adenylate Residues are added at the 3’ end.

hnRNA is fully processed into mRNA, which is then transported out of the nucleus for translation.

Genetic Code

It is the sequence of bases in mRNA that codes for a particular amino acid in the protein synthesis.

Each code is composed of three nucleotides known as a “triplet.” Codons are generally universal, with the exception of some protozoans and mitochondrial codons.

More than one triplet codon codes for the same amino acid, making the genetic code degenerate.

There are 64 codons in total, 61 of which code for amino acids.

3 codons - UAA, UAG, UGA - do not code for any amino acids and are referred to as ‘stop codons’

The start codon, as well as the amino acid methionine, is coded by AUG.

Genetic Mutation

Point Mutation: A single base pair change can result in a point mutation, such as the one that causes Sickle cell anaemia. This mutation changes a Glutamate in the normal protein to a Valine in the Sickle cell protein.

Frameshift Mutation: When one or two base pairs are either lost or gained, it causes a shift in the reading frame at the point of insertion or deletion, resulting in a frameshift mutation.

The act of changing words from one language to another

The act of translating words from one language to another

The process of amino acid polymerisation is known as translation. Peptide bonds are used to join the amino acids.

All three RNAs have a distinct role in the process of translation

1. mRNA provides the template for the sequence of amino acids in a polypeptide chain, which is determined by the sequence of bases present in mRNA.

2. tRNA acts as an adapter, connecting the mRNA codon sequence to the appropriate amino acid, and translating the genetic code.

3. rRNA - performs a structural and catalytic role

tRNA – The Adapter Molecule: Crick proposed the presence of an adapter molecule, which binds to a specific amino acid. It was initially referred to as soluble RNA (sRNA) and later named as transfer RNA (tRNA).

« The shape of tRNA is similar to an inverted ‘L’.

» There is a specific initiator tRNA that is specific for each amino acid.

» Stop codons do not have corresponding tRNAs.

» It has an anticodon loop, containing a complementary code found on mRNA.

» There is an amino acid acceptor arm which binds a specific amino acid according to the codon.

Aminoacylation of tRNA (charging of tRNA) is the first step in the process of translation.

Ribosomes are a factory for producing proteins.

The small subunit of ribosomes initiates mRNA to protein translation by hosting mRNA.

The process of translation is always in the 5’→3’ direction.

A peptide bond is formed when two amino acids present on tRNAs are in close proximity to each other.

There are two sites in the large subunit of a ribosome which can accommodate two tRNAs with amino acids close enough to form a peptide bond.

The Ribosome plays an important role as a catalyst in the formation of a peptide bond.

A ribozyme is a molecule made up of 23s rRNA, found in bacteria, that acts as an enzyme and catalyzes peptide bond formation.

The coding sequence for a polypeptide in mRNA is flanked by a start codon and a stop codon.

Untranslated Regions (UTRs) - UTRs are located at the 5’ end before the start codon and at the 3’ end after the stop codon. They are not translated, but they play an important role in making the translation process efficient.

The Release factor binds to the stop codon at the end, terminating the process and releasing the polypeptide from the ribosome.

Check Out: Difference Between CDS and ORF

![Protein Factories]()

The Central Dogma

The Central Dogma of Molecular Biology was proposed by Francis Crick, which states that genetic information flows from DNA → RNA → Protein.

Regulation of Gene Expression

Expression of a gene to form a polypeptide in eukaryotes can be regulated at various levels.

1. At the time of formation of a primary transcript (i.e. transcription)

2. During processing or splicing

3. Transport of mRNA from the nucleus to the cytosol

4. During translation, the process of protein synthesis

The expression of genes is regulated by environmental, physiological, and metabolic conditions.

The coordinated regulation and expression of several sets of genes results in the development and differentiation of an embryo.

In prokaryotes, gene expression is primarily regulated at the level of transcription initiation.

The activity of RNA polymerase at the start site is regulated by regulatory proteins, which can be either a repressor or an activator.

The accessibility of the promoter region is regulated by an operator sequence located adjacent to it, which binds with a specific protein, usually a repressor.

In each operon, there is a specific operator and repressor protein

The Lac Operon

Jacob and Monad first demonstrated the transcriptionally regulated system in the lac operon.

An operon consists of many structural genes regulated by a single promoter and regulatory gene.

The lac operon consists of

1. Regulatory Gene: Gene I (Inhibitor Gene), that codes for the repressor of the Lac Operon.

2. Structural Genes:

• Z
• Y
• A

» z gene codes for 𝜷-galactosidase, which hydrolyzes lactose into glucose and galactose.

The gene ‘y’ codes for permease, which is responsible for increasing the cell’s permeability to 𝛽-galactosides.

A gene codes for transacetylase.

The gene i continuously synthesizes a repressor which binds to the operator, thus preventing RNA polymerase from transcribing.

Lactose acts as an inducer and is a substrate of 𝜷-galactosidase, which also regulates gene expression.

When lactose or allolactose is present, it binds with the repressor, inactivating it and allowing RNA polymerase to access the promoter region, thus initiating transcription.

Negative Regulation is when a repressor is used to regulate a process.

Human Genome Project

The Human Genome Project (HGP) was launched in 1990 with the goal of deciphering the complete DNA sequence of the human genome.

Genetic engineering techniques were utilized to separate and clone the DNA segment in order to determine the DNA sequence.

The project was completed in 2003, and the sequence of chromosome 1 was finished in May 2006.

Key Findings of the Human Genome Project (HGP):

The human genome has 3164.7 million base pairs

Total approximately 30,000 genes are present with an average of 3,000 bases per gene.

The Dystrophin gene is the largest human gene, with 2.4 million bases.

99.9% of nucleotides are the same in all people

Only 2% of the genome codes for proteins

Most genes are found on chromosome 1, with 2968 being the total number of genes.

The Y chromosome has the least amount of genes, with only 231.

There are approximately 1.4 million locations where there is a single base difference in DNA, referred to as Single Nucleotide Polymorphism (SNPs) (snips).

DNA Fingerprinting

Alec Jeffreys was the creator of VNTR (Variable Number of Tandem Repeats), the technique commonly known as DNA fingerprinting.

The difference in the DNA makeup of each individual is the basis of DNA fingerprinting, and this is what accounts for the unique phenotype of each individual.

The DNA fingerprinting technique allows for the quick comparison of the DNA sequences of two individuals.

DNA fingerprinting involves identifying the difference between two DNA molecules at the specific regions where the sequence is repeated many times, referred to as repetitive DNA.

Satellite DNA, which is characterized by a small peak during density gradient centrifugation, is composed of repetitive DNAs.

Depending on the number of repetitive units, base composition, and length of the segment, satellite DNA can be further classified into microsatellites, minisatellites, etc.

These sequences do not code for protein, but they make up a significant part of the human genome.

A high degree of polymorphism present in these sequences is the basis for DNA fingerprinting

DNA fingerprinting is used for paternity tests, as this polymorphism is passed down to the child.

It has been widely utilized in forensic science.

DNA fingerprinting can be used to assess the genetic diversity within a population.