<h5 id="71-dna-as-the-genetic-material">7.1 DNA As The Genetic Material</h5> <ul> <li> <p>The nature of the gene responsible for expressing traits was a significant challenge for the scientific community.</p> </li> <li> <p>Johann Friedrich Miescher is credited with the discovery of DNA, an acidic substance he isolated from the nuclei of pus cells, which he named nuclein, containing DNA and protein.</p> </li> <li> <p>Although DNA and protein were present in the chromosome and nucleus, the nature of the genetic material remained unclear for a considerable time.</p> </li> <li> <p>Experiments with microorganisms by various investigators gradually provided evidence supporting DNA as the genetic material.</p> </li> <li> <p>DNA is responsible for determining the trait or feature of any organism, with a few exceptions of some viruses.</p> </li> <li> <p>Therefore, DNA is the primary chemical component in the chromosome that serves as the genetic material.</p> </li> </ul> <p>======</p> <h5 id="711-discovery-of-the-transforming-principle">7.1.1 Discovery of the transforming principle</h5> <ul> <li>Frederick Griffith discovered two different strains of Streptococcus pneumoniae bacteria: virulent (S) with a polysaccharide capsule and non-virulent (R) without a capsule.</li> <li>Injecting live S bacteria into mice caused pneumonia and death, while R bacteria had no ill effects on mice.</li> <li>Heat-killing S bacteria and injecting them into mice did not harm the mice, but a mixture of heat-killed S and live R bacteria resulted in mice dying from pneumonia due to the presence of live S bacteria.</li> <li>Griffith concluded that R-strain bacteria must have taken up a “transforming principle” from heat-killed S bacteria, allowing them to transform into smooth-coated bacteria and become virulent.</li> <li>The transforming substance was later identified as DNA, marking the discovery of the transforming principle and the process of transformation.</li> </ul> <p>======</p> <h5 id="712-biochemical-characterisation-of-transforming-principle">7.1.2 Biochemical characterisation of transforming principle</h5> <ul> <li>Avery, Macleod, and McCarty conducted experiments to identify the Griffith’s transforming principle.</li> <li>They prepared an extract of heat-killed smooth strain of bacteria and treated it with ribonuclease (RNase), deoxyribonuclease (DNase), and protease.</li> <li>Transformation of rough strain into the smooth strain was observed in those colonies in which RNase and protease treated extract were added, but not in the colony to which DNase treated extract was added.</li> <li>The results confirmed that DNA is the transforming principle.</li> <li>This discovery supported the hypothesis that the genetic material of the cells is either DNA or RNA, and the experiment concluded that transformation requires DNA, making DNA the genetic material of the cell.</li> </ul> <p>======</p> <h5 id="713-the-hershey---chase-experiment">7.1.3 The Hershey - Chase experiment</h5> <ul> <li> <p>The Hershey-Chase experiment (1952) provided evidence supporting DNA as genetic material using T2 bacteriophages and E. coli.</p> </li> <li> <p>T2 bacteriophages, grown in radioactive phosphorus (${ }^{32}\mathrm{P}$) and radioactive sulfur ($\left({ }^{35} \mathrm{S}\right)$) media, were used to infect separate cultures of unlabelled E. coli.</p> </li> <li> <p>After infection, the bacterial colonies were agitated in a blender, centrifuged, and separated into pellets (bacteria) and supernatant (phage debris).</p> </li> <li> <p>Results showed that pellets infected with ${ }^{32}\mathrm{P}$ bacteriophages had radioactivity, while supernatant infected with ${ }^{35} \mathrm{S}$ bacteriophages did not, indicating DNA entered the bacteria, not proteins.</p> </li> <li> <p>This led to the conclusion that DNA is the genetic material, although its role as a repository of genetic information was not yet clear.</p> </li> <li> <p>Subsequent studies, including the discovery of DNA structure (Chargaff, Wilkins, Franklin, Watson, and Crick), clarified how DNA can encode large amounts of information.</p> </li> </ul> <p>======</p> <h5 id="72-prokaryotic-and-eukaryotic-gene-organisation">7.2 Prokaryotic and Eukaryotic Gene Organisation</h5> <ul> <li>Genes are units of inheritance that control specific traits or characters, and are made up of a segment of DNA that expresses itself through the synthesis of a polypeptide chain via RNA synthesis, also known as the ‘Central dogma’ of molecular biology.</li> <li>The relationship between genes and enzymes was established through experiments on the mould Neurospora crassa, which showed that genes control the synthesis of specific enzymes.</li> <li>The discovery of chromatin fibre through electron microscopy revealed a bead on a string like structure, with each bead representing a gene. However, further investigations showed that each bead is a nucleosome, and a gene is a segment of DNA with a specific promoter region where RNA polymerase can bind and transcribe mRNA.</li> <li>The total DNA content in one complete set of chromosomes, including all the genes and other parts of DNA, is called a genome. Viral and bacterial genomes are much smaller than eukaryotic genomes, which are more complex and can contain a large amount of non-coding sequence.</li> <li>In eukaryotic gene expression, the size of active mRNA involved in the process of polypeptide chain synthesis is much smaller than the primary transcript, due to the process of splicing where interspersed segments of the primary transcript are excised and remaining portions are joined together to form mRNA.</li> </ul> <p>======</p> <h5 id="nuclear-genome">Nuclear Genome</h5> <ul> <li>The majority of DNA is found in the nucleus and is referred to as nuclear DNA.</li> <li>In prokaryotes, the genome is mostly composed of coding DNA sequences. In contrast, eukaryotic genomes have a small proportion of coding regions, making up approximately 2% of the total genome.</li> <li>The human genome, for example, is about 3,000Mb or 3 billion base pairs of DNA, containing an estimated 20,000 genes. There are also non-coding regions in the genome, which contain sequences repeated thousands to millions of times in tandem arrays.</li> <li>The size and number of these repetitive DNA sequences vary significantly in different genomes.</li> <li>The nuclear genome in a eukaryotic cell is organized into smaller, condensed units called chromosomes, which are composed of linear DNA molecules. A complete set of chromosomes in a haploid genome is represented by the letter n, while most organisms carry two sets of chromosomes in each cell, known as diploid organisms (2n).</li> </ul> <p>======</p> <h5 id="organellar-genomes">Organellar Genomes</h5> <ul> <li>Organellar genomes are found in certain membrane-bound cellular organelles, such as chloroplasts and mitochondria.</li> <li>They are primarily circular, double-stranded DNA and are present in multiple copies within each organelle.</li> <li>Organelle genomes replicate in a semi-conservative manner and are inherited independently of the nuclear genome.</li> <li>The DNA in organelles contains genes essential for organelle-specific functions.</li> <li>Organelle-DNA is usually uniparental, meaning it is inherited from only one parent, and is typically passed on to the next generation through female gametes.</li> </ul> <p>======</p> <h5 id="73-dna-replication">7.3 DNA Replication</h5> <ul> <li>In 1953, Watson and Crick proposed that DNA replication occurs via a semiconservative mechanism, based on the complementary relationship of bases of two polynucleotide chains.</li> <li>During replication, the two strands of DNA separate, and each strand serves as a template for the synthesis of a new complementary strand due to specific base pairing (A-T and C-G).</li> <li>This results in the formation of two identical daughter duplexes, each consisting of one parental strand and one newly synthesized daughter strand.</li> <li>The semiconservative replication model was supported by the evidence provided by Matthew Messelson and Franklin Stahl in 1958.</li> <li>In this mode of replication, the parental duplex DNA separates into two daughter duplexes, each containing one original (parental) strand and one newly synthesized strand.</li> </ul> <p>======</p> <h5 id="731-messelson-and-stahl-experiment">7.3.1 Messelson and Stahl experiment</h5> <ul> <li>Messelson and Stahl used two isotopes of nitrogen, ${ }^{14} \mathrm{~N}$ and ${ }^{15}\mathrm{~N}$, to distinguish between old and new strands of DNA in E. coli.</li> <li>They observed that DNA extracted from E. coli grown in ${ }^{15} \mathrm{~N}$ produced a single band at the lower side of the centrifuge tube, while DNA extracted from bacterial cells grown in ${ }^{14} \mathrm{~N}$ formed a band closer to the top.</li> <li>When E. coli bacteria were transferred from ${ }^{15}\mathrm{~N}$ medium to ${ }^{14} \mathrm{~N}$ medium, the DNA extracted from the bacteria after the first round of division produced a single band at a position intermediate between the bands of heavy DNA (${ }^{15}\mathrm{~N}$) and light DNA (${ }^{14} \mathrm{~N}$).</li> <li>After a second round of replication in ${ }^{14} \mathrm{~N}$ medium, two bands of equal intensity appeared in the centrifuge tube, one in the intermediate position and the other at the position expected of DNA that contained only ${ }^{14} \mathrm{~N}$ DNA.</li> <li>Density gradient centrifugation is a centrifugation technique used to separate molecules based on their density, and it was used in this experiment to separate DNA based on the density of the nitrogen isotopes.</li> </ul> <p>======</p> <h5 id="732-interpretation-of-results-by-messelson-and-stahl">7.3.2 Interpretation of results by Messelson and Stahl</h5> <ul> <li>After the first replication round in Messelson and Stahl’s experiment, each daughter DNA duplex was a hybrid with one heavy strand containing ${ }^{15} \mathrm{~N}$ from the parent and one light strand containing ${ }^{14} \mathrm{~N}$ from the medium.</li> <li>In the second replication round, the heavy template strand formed another hybrid duplex DNA, while the lighter strand formed a light DNA duplex.</li> <li>This result confirmed the semiconservative replication prediction of the Watson and Crick model, in which the original DNA molecule serves as a template for the synthesis of a new complementary strand.</li> <li>Taylor and colleagues in 1958 conducted similar experiments in Vicia faba using radioactive thymidine, confirming that DNA in chromosomes replicates semiconservatively.</li> <li>Both experiments provided crucial evidence supporting the semiconservative DNA replication mechanism.</li> </ul> <p>======</p> <h5 id="733-the-machinery-of-replication">7.3.3 The machinery of replication</h5> <ul> <li>Replication of double stranded DNA molecule requires unwinding for separating the strands and exposing the bases.</li> <li>Both DNA strands serve as templates for the assembly of complementary bases to form new polynucleotide strands which are anti-parallel to the template strand.</li> <li>Several proteins and enzymes are involved in the replication of DNA in both prokaryotes and eukaryotes, including: <ul> <li>Helicase: Unwinds the double helix</li> <li>Single-stranded binding proteins: Prevent the unwound strands from rewinding</li> <li>Primase: Synthesizes short RNA segments to provide a starting point for DNA polymerase</li> <li>DNA polymerase: Synthesizes new strands of DNA</li> </ul> </li> <li>DNA polymerase III is the main enzyme responsible for DNA replication in prokaryotes.</li> <li>DNA polymerase α, β, and γ are the main enzymes responsible for DNA replication in eukaryotes.</li> </ul> <p>======</p> <h5 id="i-dna-polymerases">(i) DNA Polymerases</h5> <ul> <li>DNA Polymerases are the main enzymes of replication, responsible for synthesizing new DNA strands in 5’→3’ direction.</li> <li>In prokaryotes, there are three kinds of DNA Polymerases: I, II, and III, with DNA Polymerase III being the main enzyme for DNA replication.</li> <li>DNA Polymerase III synthesizes new strands in 5’→3’ direction and has 3’→5’ exonuclease activity for error correction.</li> <li>DNA Polymerase I has 5’→3’ polymerisation activity and both 5’→3’ and 3’→5’ exonuclease activity, used for removing RNA primers laid down by primase.</li> <li>DNA Polymerase II is involved in DNA repair, while multiple DNA Polymerases in eukaryotes handle synthesis and repair of DNA.</li> </ul> <p>======</p> <h5 id="ii-primase">(ii) Primase</h5> <ul> <li>DNA polymerase is unable to begin synthesis of a new DNA strand.</li> <li>A RNA primer, providing a $3^{’}\mathrm{OH}$ group for DNA polymerase to add new nucleotides, is required by DNA polymerase.</li> <li>Primase, a DNA-dependent RNA polymerase, is responsible for the synthesis of RNA primers.</li> <li>RNA primers are short RNA oligonucleotide chains, approximately 10-12 nucleotides long.</li> <li>Primase synthesizes RNA primers to initiate DNA replication.</li> </ul> <p>======</p> <h5 id="iii-helicases">(iii) Helicases</h5> <ul> <li>DNA helicases are enzymes that help in the unwinding of the DNA double helix.</li> <li>They break hydrogen bonds between the bases of two strands of a DNA molecule.</li> <li>This process is necessary for generating single strand templates.</li> <li>Helicases use energy in the form of ATP to carry out this function.</li> <li>The breaking of hydrogen bonds by helicases results in the separation of the two strands of the DNA molecule.</li> </ul> <p>======</p> <h5 id="iv-topoisomerase">(iv) Topoisomerase</h5> <ul> <li>Topoisomerases are enzymes that relieve the torsional strain caused by the separation of the two strands of DNA.</li> <li>They do this by nicking and religating the DNA, which helps in maintaining the coiled and supercoiled structure of the DNA helix.</li> <li>The function of topoisomerases becomes particularly important during DNA replication, as the replication fork can cause torsional strain.</li> <li>Topoisomerases help in preventing the formation of knots in the DNA helix in front of the replication fork.</li> <li>They are essential for the proper functioning of the DNA replication process and for maintaining the structural integrity of the DNA.</li> </ul> <p>======</p> <h5 id="v-single-strand-binding-ssb-proteins">(v) Single strand binding (SSB) proteins</h5> <ul> <li>Single strand binding (SSB) proteins prevent the reannealing of single stranded DNA.</li> <li>After helicase unwinds DNA, the single stranded nucleotide chains are susceptible to forming hydrogen bonds again.</li> <li>SSB proteins attach tightly to these exposed single stranded DNA molecules.</li> <li>This attachment of SSB proteins keeps the DNA in single stranded form.</li> <li>This is essential for DNA replication to occur.</li> </ul> <p>======</p> <h5 id="vi-dna-ligase">(vi) DNA ligase</h5> <ul> <li>DNA ligase is an enzyme that aids in the joining of newly synthesized DNA fragments.</li> <li>It forms a phosphodiester bond between the $3^{\prime} \mathrm{OH}$ group and the 5’ phosphate group.</li> <li>This process is crucial for DNA replication and repair.</li> <li>DNA ligase plays a key role in maintaining the integrity of DNA molecules.</li> <li>The enzyme is also essential for the formation of circular DNA molecules, as in plasmids and viruses.</li> </ul> <p>======</p> <h5 id="734-mechanism-of-dna-replication-in-bacteria">7.3.4 Mechanism of DNA replication in bacteria</h5> <ul> <li>DNA replication in bacteria begins at a specific site called the origin of replication (ori).</li> <li>Bacterial chromosomes are circular and double-stranded, with a single replication origin and a terminus. Helicase enzymes unwind the DNA strands, and single strand binding proteins stabilize the single-stranded DNA.</li> <li>DNA polymerase III synthesizes new DNA strands in the 5’ o 3’ direction, while primase synthesizes RNA primers for DNA synthesis.</li> <li>Replication occurs in opposite directions from the origin, with leading and lagging strands formed. The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously in short fragments called Okazaki fragments.</li> <li>DNA polymerase I removes RNA primers and replaces them with DNA nucleotides, and DNA ligase joins the Okazaki fragments together. Replication is terminated when two replication forks meet.</li> </ul> <p>======</p> <h5 id="74-gene-expression">7.4 Gene Expression</h5> <ul> <li>Gene expression is the process by which the information in a gene is converted into a functional gene product, usually a protein.</li> <li>The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins in a unidirectional manner.</li> <li>Transcription is the first step in gene expression where genetic information is transferred from DNA to mRNA.</li> <li>In translation, the information is transferred from mRNA to a polypeptide chain.</li> <li>In eukaryotes, mRNA serves as an intermediate between a gene present in DNA and the peptide it encodes, allowing for amplification and movement into the cytoplasm for protein synthesis.</li> <li>Reverse transcription is a process occurring in some viruses like retroviruses, where the genetic information flows from RNA to DNA, reversing the central dogma.</li> </ul> <p>======</p> <h5 id="741-transcription">7.4.1 Transcription</h5> <ul> <li>All cellular RNAs (mRNA, tRNA, rRNA) are synthesized on a DNA template in a process called transcription.</li> <li>Transcription is similar to replication but differs in that only small parts of DNA are copied into RNA, not the entire template.</li> <li>The DNA strand with a base sequence identical to the transcribed RNA (except for T to U substitution) is called the sense or coding strand.</li> <li>The other strand, used as a template for RNA synthesis, is called the template or non-coding strand.</li> <li>Only one of the DNA strands is typically transcribed into RNA within a gene.</li> </ul> <p>======</p> <h5 id="742-rna-polymerase">7.4.2 RNA polymerase</h5> <ul> <li>RNA polymerase is the enzyme responsible for transcription in both prokaryotes and eukaryotes.</li> <li>In prokaryotes, there is a single type of RNA polymerase for all types of RNA (mRNA, rRNA, and tRNA).</li> <li>In eukaryotes, three distinct types of RNA polymerases are present: RNA polymerase I transcribes 28S, 18S and 5.8S rRNA; RNA polymerase II transcribes hnRNA (precursor of mRNA); and RNA polymerase III transcribes tRNA and 5S rRNA.</li> <li>RNA polymerase enzyme does not require a primer and synthesizes new chain in 5’ → 3’ direction using ribonucleoside triphosphates (rNTPs) as substrates.</li> <li>The prokaryotic RNA polymerase is a large enzyme with two a, one β, and one β’ subunits, forming the core enzyme (α2, β, β’). When associated with a sigma factor, it becomes the holoenzyme (α2, β, β’, σ).</li> </ul> <p>======</p> <h5 id="743-transcription-unit">7.4.3 Transcription unit</h5> <ul> <li>A transcription unit is a stretch of DNA where RNA is transcribed.</li> <li>RNA polymerase recognizes a transcription unit through a promoter, a DNA sequence upstream of the start site.</li> <li>The promoter serves as the binding site for the RNA polymerase and indicates the start location and direction of synthesis.</li> <li>In bacteria, the promoter often contains a consensus sequence called the Pribnow box (TATAAT), where initial melting of duplex DNA occurs.</li> <li>Transcription begins at the start site and ends at the terminator site, marking the end of the transcription unit.</li> </ul> <p>======</p> <h5 id="744-initiation">7.4.4 Initiation</h5> <ul> <li>Transcription initiation involves RNA polymerase binding to the promoter.</li> <li>In bacteria, the sigma subunit of RNA polymerase recognizes and binds to the promoter.</li> <li>RNA polymerase unwinds the DNA helix at the TATAAT sequence, creating a single-stranded DNA template.</li> <li>RNA synthesis begins with RNA polymerase pairing a ribonucleoside triphosphate to the complementary base on the DNA template strand.</li> <li>The sigma subunit is typically released after initiation, and the RNA polymerase holoenzyme continues RNA synthesis.</li> </ul> <p>======</p> <h5 id="745-elongation">7.4.5 Elongation</h5> <ul> <li>The region where RNA polymerase, DNA, and growing RNA transcript are located is called a transcription bubble due to the presence of a locally unwound ‘bubble’ of DNA.</li> <li>RNA polymerase moves along the template strand, unwinds DNA at the leading edge of the transcription bubble, and joins nucleotides to the 3’OH end of the RNA molecule according to the sequence of the template.</li> <li>As the RNA chain elongation continues, the new RNA molecule separates from the DNA template, and the DNA double helix reforms behind the transcription bubble.</li> <li>The rate of transcription in bacterial cells at 37°C is approximately 40 nucleotides per second.</li> <li>During elongation, RNA polymerase adds rNTP to the 3’OH end of the growing polynucleotide chain.</li> </ul> <p>======</p> <h5 id="746-termination">7.4.6 Termination</h5> <ul> <li>The elongation of RNA chain stops when RNA polymerase reaches the terminator sequence in DNA.</li> <li>The RNA-DNA hybrid helix within the transcription bubble dissociates, and RNA polymerase separates from template DNA.</li> <li>The two strands of DNA rewind and the newly synthesized RNA chain gets released.</li> <li>In prokaryotic transcription, termination can be rho protein-dependent (rho-dependent termination) or rho-independent.</li> <li>In prokaryotes, a group of genes can be transcribed into a single RNA molecule, called polycistronic RNA, when a single termination sequence is present at the end of several genes that are transcribed together.</li> <li>Translation can begin before the RNA is fully transcribed since mRNA does not require processing and transcription and translation occur in the cytoplasm.</li> </ul> <p>======</p> <h5 id="747-transcription-in-eukaryotes">7.4.7 Transcription in eukaryotes</h5> <ul> <li>The mechanism of transcription by RNA polymerase in eukaryotes is similar to that in prokaryotes, but with additional steps and factors.</li> <li>Eukaryotic transcription requires accessory factors called transcription factors for proper initiation by RNA polymerase I, II, and III at promoters of rRNA, mRNA, and tRNA genes.</li> <li>Most eukaryotic genes are split genes, containing introns (non-coding regions) and exons (coding regions).</li> <li>Primary transcripts, called heterogenous nuclear RNA (hnRNA), contain both exons and introns and undergo post-transcriptional modifications before being transferred into the cytoplasm from the nucleus.</li> <li>Post-transcriptional modifications include capping, removal of introns (splicing), and addition of a poly-A tail.</li> </ul> <p>======</p> <h5 id="_1-capping_"><em>1. Capping</em></h5> <ul> <li>The 5’ end of eukaryotic pre-mRNA has a purine (A or G).</li> <li>A GTP called a 5’ cap is added to the 5’ end of pre-mRNA.</li> <li>The guanine in the GTP is modified by the addition of a methylgroup, known as the 5’ methyl G cap.</li> <li>This cap formation protects the 5’ end of mRNA from degradation by exonuclease.</li> </ul> <p>======</p> <h5 id="2-_splicing_">2. <em>Splicing</em></h5> <ul> <li>Splicing is a process in the creation of mature mRNA.</li> <li>The primary transcript or pre-mRNA contains both exons and introns.</li> <li>Introns are removed while exons are joined together during this process.</li> <li>This results in a continuous stretch of exons that forms the mature mRNA.</li> <li>The process of splicing is essential for correct protein synthesis.</li> </ul> <p>======</p> <h5 id="3-_poly-a-tail_">3. <em>Poly-A tail</em></h5> <ul> <li>The poly-A tail, also known as poly-adenylated tail, is added to the 3’ end of pre-mRNA after transcription.</li> <li>This tail is a series of adenine nucleotides.</li> <li>The poly-A tail aids in the stability of mRNA.</li> <li>It does so by protecting mRNA from degradation.</li> <li>The length of the poly-A tail can influence the stability and translation efficiency of mRNA.</li> </ul> <p>======</p> <h5 id="75-genetic-code">7.5 Genetic Code</h5> <ul> <li>The genetic code is the set of rules that pairs each codon (a sequence of three DNA or RNA nucleotides) with an amino acid, or a stop signal to end protein synthesis.</li> <li>There are 64 codons, 61 of which code for 20 amino acids, while the remaining three (UAA, UAG, UGA) are stop codons.</li> <li>AUG, which codes for methionine, has a dual function as it also acts as an initiator codon.</li> <li>The genetic code is unambiguous (one codon codes for only one amino acid) and degenerate (each amino acid may be specified by more than one codon), except for methionine and tryptophan, which are encoded by a single codon each.</li> <li>The genetic code is non-overlapping, meaning each base along the mRNA is a part of only one codon.</li> <li>The genetic code is universal, with some exceptions found in mitochondrial codons and some protozoans.</li> </ul> <p>======</p> <h5 id="76-translation">7.6 Translation</h5> <ul> <li>Translation is the process of synthesizing a polypeptide chain on an mRNA bound to ribosomes.</li> <li>The four steps of translation are: (i) charging of tRNA, (ii) initiation of translation, (iii) elongation, and (iv) termination.</li> <li>Genetic information from DNA is first transcribed into mRNA, which carries the information in the form of codons for the sequence of amino acids in specific polypeptide chains.</li> <li>In ribosomes, the information present in the language of codons is decoded into the language of amino acids, which join to form a polypeptide chain.</li> <li>The charging of tRNA involves the attachment of specific amino acids to their corresponding tRNA molecules.</li> </ul> <p>======</p> <h5 id="charging-of-trna">Charging of tRNA</h5> <ul> <li>tRNA molecules deliver amino acids to ribosomes for protein synthesis.</li> <li>The anticodon region of tRNA recognizes and base pairs with specific mRNA codons through hydrogen bonding.</li> <li>One tRNA molecule’s anticodon can recognize multiple mRNA codons due to wobble pairing, where the base pairing is precise at the first two positions and flexible at the third base of the codon.</li> <li>Amino acids are first activated by aminoacyl-tRNA synthetase and then transferred to their specific tRNA molecules.</li> <li>The charging of tRNA involves two steps: (1) amino acid reacts with ATP and aminoacyl-tRNA synthetase, producing aminoacyl-AMP and PPi, and (2) aminoacyl-AMP reacts with tRNA, producing aminoacyl-tRNA and AMP. The activated amino acid is attached to the 3’OH end of the tRNA (CCA) sequence.</li> </ul> <p>======</p> <h5 id="initiation-of-translation">Initiation of Translation</h5> <ul> <li>Ribosomes have three sites for tRNA binding: A site, P site, and E site.</li> <li>During initiation, the 30S subunit of prokaryotic ribosomes binds to the ribosome binding sequence at the $5^{\prime}$ end of mRNA.</li> <li>In prokaryotes, the initiating aminoacyl-tRNA or fMet-tRNA$^{fmet}$ attaches to the initiation codon AUG at the P site.</li> <li>The 50S subunit then associates with the 30S subunit to form the 70S initiation complex, aided by initiation factors and GTP.</li> <li>In eukaryotes, the small subunit of ribosomes (40S) recognizes the cap and binds to it, then moves along the mRNA until it locates the initiating AUG codon.</li> </ul> <p>======</p> <h5 id="elongation">Elongation</h5> <ul> <li>Elongation is a step in protein synthesis where amino acids are joined to create a polypeptide chain.</li> <li>The initiation complex has P site occupied by amino acyl-tRNA with formylated methionine (fMet-tRNA${ }^{\text{fmet})}$ in prokaryotes and methionine (Met-tRNA ${ }^{\text {met) }}$ ) in eukaryotes.</li> <li>A peptide bond is formed between the amino acids at P and A sites by peptidyl transferase enzyme, releasing the amino acid in the P site from its tRNA.</li> <li>The ribosome moves along mRNA in 5’ → 3’ direction, bringing the uncharged tRNA from P site to E site and the peptidyl-tRNA with growing polypeptide chain from A site to P site.</li> <li>Several proteins factors and GTP are involved in the elongation step, with the A site now ready to receive the next aminoacyl-tRNA molecule specified by the codon.</li> </ul> <p>======</p> <h5 id="termination">Termination</h5> <ul> <li>The elongation of the polypeptide chain continues until a stop codon on the mRNA enters the A site of the ribosome.</li> <li>The three stop codons are UAA, UAG, and UGA, which do not code for any amino acid.</li> <li>No tRNA with an amino acid enters into the A site of the ribosome when a termination codon occupies it.</li> <li>Release factors recognize stop codons and bind to the A site, releasing the polypeptide chain from tRNA in the P site.</li> <li>Other protein factors bring about the release of tRNA, mRNA, and finally the dissociation of the ribosome.</li> <li>Proteins synthesized in ribosomes undergo post-translational modifications (PTMs) to form the mature protein product, which comes in a wide variety of types and are mostly catalyzed by enzymes that recognize specific target sequences in specific proteins.</li> </ul> <p>======</p> <h5 id="polyribosomes">Polyribosomes</h5> <ul> <li>Polyribosomes are clusters of ribosomes attached to a single mRNA molecule.</li> <li>They are responsible for simultaneous translation of a single mRNA into multiple polypeptide chains.</li> <li>This process increases the efficiency and speed of protein synthesis.</li> <li>Polyribosomes are found in both prokaryotes and eukaryotes.</li> <li>The first ribosome translating the mRNA must move far enough past the start codon to allow a second ribosome to attach, resulting in the formation of polyribosomes.</li> </ul> <p>======</p> <h5 id="77-gene-mutation">7.7 Gene Mutation</h5> <ul> <li> <p>Mutation is a sudden change in the genetic material (DNA or chromosome) that can occur in both somatic and germinal cells.</p> </li> <li> <p>Mutations in germinal cells that get inherited to offspring have significance, while those in somatic cells do not.</p> </li> <li> <p>Mutations in chromosomes can be structural or numerical, leading to chromosomal aberrations or ploidy changes, respectively.</p> </li> <li> <p>Mutations at the molecular level, specifically in genes, can result from errors during replication or from external mutagens.</p> </li> <li> <p>Gene mutations can be categorized into addition, deletion, and substitution of one or a few nucleotides, with the latter two altering the reading frame and potentially affecting the resulting protein.</p> </li> <li> <p>Substitution mutations can be further classified into transition and transversion, depending on the replaced nucleotides.</p> </li> </ul> <p>======</p> <h5 id="771-molecular-mechanism-of-mutation">7.7.1 Molecular mechanism of mutation</h5> <ul> <li> <p>Spontaneous mutations can occur due to tautomeric shifts in the nitrogenous bases of DNA, leading to a temporary enol or imino form that pairs with a different nucleotide. This can result in a substitution of the $G o C$ pair with an $A = T$ pair.</p> </li> <li> <p>Induced mutations can be caused by physical mutagens like X-rays or UV rays, or chemical mutagens like alkylating agents or base analogs.</p> </li> <li> <p>Physical mutagens like X-rays can cause breakage of bonds in the DNA molecule, leading to deletion of nucleotides and frame shift mutations. Non-ionising radiation like UV rays can excite electrons in the DNA molecule, leading to deletion or substitution mutations.</p> </li> <li> <p>Chemical mutagens like 5-bromouracil can get incorporated into the DNA as a complementary base against adenine, but can later pair with guanine nucleotide due to tautomeric shifts, resulting in substitution mutations.</p> </li> <li> <p>Alkylating agents like EMS ethylate DNA nucleotides, altering their pairing properties and leading to substitution mutations. Many chemical mutagens induce mutations through altered pairing properties.</p> </li> </ul> <p>======</p> <h5 id="78-dna-repair">7.8 DNA Repair</h5> <ul> <li>DNA repair is a mechanism that corrects most errors that occur during DNA replication.</li> <li>Excision repair is a type of DNA repair in which altered or modified bases are removed or excised. Base excision repair is a mechanism that involves recognition of altered bases by enzyme DNA glycosylase, which is followed by removal of the altered base and filling of the gap by a DNA polymerase enzyme.</li> <li>Nucleotide excision repair is another type of excision repair that repairs damage formed by thymine dimer. This mechanism recognizes and binds to the dimer site, bends it, and removes a portion of the DNA strand having the damaged dimer. The gap is then filled by DNA polymerase I and sealed by DNA ligase.</li> <li>Mismatch repair is a mechanism that corrects wrong nucleotides incorporated during DNA replication. It recognizes mismatch by MutS and subsequently binds MutH and MutL proteins, forming a complex that breaks the strand and excises the nicked portion including the mismatch. The gap is then filled by DNA polymerase and joined by DNA ligase.</li> <li>Other DNA repair mechanisms have been observed and studied in both prokaryotes and eukaryotes, which can be studied in higher classes. The stability of genetic information can be appreciated based on the account of mutation and repair mechanisms.</li> </ul> <p>======</p> <h5 id="79-recombination">7.9 Recombination</h5> <ul> <li>Recombination was observed by geneticists soon after the rediscovery of Mendel’s principles in 1900.</li> <li>The phenomenon of linkage was established, where genes on the same chromosome inherit together, leading to deviation from the 9:3:3:1 ratio in dihybrid crosses.</li> <li>Recombinants are due to crossing over, where exchange of parts of homologous chromosomes during meiosis results in new combinations of genes.</li> <li>The frequency of recombinants helps in preparing a physical map of the chromosome, with 1% of recombinants between two traits being 1 map unit or 1 centimorgan apart.</li> <li>Experiments by Harriet Creighton and Barbara McClintock (1931) in maize provided evidence that crossing over is responsible for recombination, as recombinants had different morphological forms of chromosome 9.</li> </ul> <p>======</p> <h5 id="710-regulation-of-gene-expression">7.10 Regulation of Gene Expression</h5> <ul> <li> <p>In multicellular organisms, all cells have identical genes due to mitotic divisions from a zygote, but not all genes are expressed in all cells. The ‘switching on and off’ mechanism of gene action is known as regulation of gene expression.</p> </li> <li> <p>This mechanism allows specific genes to be expressed at specific times according to the cell’s needs. For example, E. coli express different genes for breaking down different sugars like glucose or lactose.</p> </li> <li> <p>Regulation of gene expression can occur at various steps along the pathway from genotype to phenotype, including chromatin level, transcription level, mRNA processing (in eukaryotes), transport of mRNA, and translational level.</p> </li> <li> <p>Transcription initiation is a crucial point of gene regulation, where the cell decides which gene to express and to what degree. Some genes are expressed at a constant level and are called ‘housekeeping genes’ or ‘constitutive genes’.</p> </li> <li> <p>The rate of expression of most genes, however, alters in cells according to the molecular signal it receives. This type of control is called regulated gene expression, where the product level of these genes rises and falls according to the cell’s needs.</p> </li> </ul> <p>======</p> <h5 id="7101-regulation-of-gene-expression-in-bacteria">7.10.1 Regulation of gene expression in bacteria</h5> <ul> <li>In bacteria, genes that have related functions are clustered and often transcribed together into a single mRNA molecule, called an operon.</li> <li>An operon typically has a set of structural genes or cistrons at one end, which are transcribed and then translated to produce different proteins.</li> <li>The regulation of operon was first described by Francois Jacob and Jacque Monod in 1961 and proposed the ‘operon model’ for the genetic control of lactose metabolism in E. coli cells.</li> <li>There are two types of operon: inducible operons and repressible operons, based on the nature of their response to an effector molecule.</li> <li>The regulation of gene expression in bacteria is controlled by two types of transcriptional control: negative control, where the regulatory protein is a repressor that inhibits transcription, and positive control, where the regulator protein is an activator which stimulates transcription.</li> </ul> <p>======</p> <h5 id="7102-the-lac-operon---an-inducible-operon">7.10.2 The Lac operon - an inducible operon</h5> <ul> <li>The lac operon in $E$. <em>coli</em> is an inducible operon that regulates the metabolism of lactose.</li> <li>The lac operon consists of three structural genes: lacZ, lacY, and lacA, which encode β-galactosidase, β-galactoside permease, and β-galactoside transacetylase, respectively.</li> <li>The regulator gene lacI, located upstream of the lac operon, encodes a repressor protein that binds to the operator of the operon and prevents transcription of structural genes in the absence of lactose.</li> <li>In the presence of lactose, β-galactosidase converts some of it into allolactose, which acts as an inducer and binds to the active repressor, causing a conformational change that renders it inactive.</li> <li>The inactive repressor fails to bind to the operator, allowing RNA polymerase to bind and transcribe lacZ, lacY, and lacA into a polycistronic mRNA, which translates into three different enzymes required for lactose metabolism.</li> </ul> <p>======</p>