LESSON 3: THE CENTRAL DOGMA INTRODUCTION Your DNA, or deoxyribonucleic acid, contains the genes that determine who you are. How can this organic molecule control your characteristics? DNA contains instructions for all the proteins your body makes. Proteins, in tum, determine the structure and function of all your cells. What determines a protein's structure? It begins with the sequence of amino acids that make up the protein. Instructions for making proteins with the correct sequence of amino acids are ‘encoded in DNA. 1. THE CENTRAL DOGMA DNA is found in chromosomes. In eukaryotic cells, chromosomes always remain in the nucleus, but proteins are made at ribosomes in the cytoplasm. How do the instructions in DNA get to the site of protein synthesis outside the nucleus? Another type of nucleic acid is responsible. This nucleic acid is RNA or ribonucleic acid. RNA is a small molecule that can squeeze through pores in the nuclear membrane. It carries the information from DNA in the nucleus to a ribosome in the cytoplasm and then helps assemble the protein. In short: Discovering this sequence of events was a major milestone in molecular biology. It is called the central dogma of molecular biology. Sometimes, when we talk about people's, appearances, special skills or behaviors, we say, ‘It's in your genes.’ It's true: the genetic information carried in your DNA sequence determines many things about you, such as the color of your hair, the shape of your eyes, your blood type, and even your susceptibility to certain diseases. But, did you ever wonder how a particular sequence of nucleotides, called A, C, G, and T, can lead to noticeable physical characteristics? The central dogma of biology describes just that. It provides the basic framework for how genetic information flows from a DNA sequence to a protein produced inside cells. This process of genetic information flowing from DNA to RNA to protein is called gene expression. Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation. However, prior to that, DNA first undergoes a process called replication. In this module, we will discuss the detailed step-by-step process of the central dogma. 1.1 REPLICATION OF DNA DNA (Deoxyribonucleic Acid) is the genetic material of all organisms on Earth from microbes to plants and human beings. An organism's complete set of DNAs, including all of its genes is called the genome. A genome contains a complete set of information that determines inherited physical characteristics such as height, skin, eye and hair color, and many others. Every cell in the human body nearly has similar DNA and in eukaryoticcells (cells that contain a nucleus and organelles, and are enclosed by a plasma membrane). DNA is a thin long molecule found in the cell's nucleus which is made up of nucleotides. The basic structure of nucleotides consists of a phosphate group, sugar and a nitrogenous base which will be further discussed in the next lessons. The four different type of nucleotides of DNA are adenine, thymine, guanine and cytosine which are represented by their first letter A, T, G, C, These four nucleotides are paired as (Adenine-Thymine) and (Guanine-Cytosine) into billions to organize a double helix structure. Figure 1. liustration to show the structure of DNA double helix DNA molecules fold into paired packages called chromosomes that are stored in the nucleus of the cell. Different species have different numbers of chromosomes, and humans have 23 pairs. Chromosomes contain many genes and on each string of DNA contains the gene which is the basic unit of heredity and a segment that describes how a certain protein is made. ‘remerome V7 e Figure 2. Illustration to show that a cell contains the genome, chromosomes, DNA and genes. James Watson and Francis Crick in 1953, worked out that DNA is a double helix that appears like a staircase. The sides of the double helix structure are the sugar-phosphate backbones and the steps or rungs are the base pairs. DNA Replication is the process of DNA duplication from an existing DNA. The replication of DNA is important for the growth repair and reproduction of cells of an organism. This process‘ocours in the nucleus of eukaryotic cells before a cell divides either by mitosis or meiosis, When a cell divides, each resulting cell keeps a copy of all of its chromosomes. DNA replication is a semi-conservative process, because when a new double-stranded DNA molecule is formed: One strand will be from the original template molecule, One strand will be newly synthesized. The two DNA strands are anti-parallel. It means that they run in opposite directions. 5 and 3' refers to the number of carbon on the pentose sugar to which P or OH is attached The major key players in DNA replication are the enzymes helicase, primase, DNA polymerase and ligase. Helicase is the unzipping enzyme that unzips the two strands of DNA in the double helix through the hydrogen bond that holds the two base pairs together. Primase will initialize the process and direct the DNA polymerase for it to figure out where it gets to start. This, primer is the starting point for DNA synthesis, The primers are made of RNA (Ribonucleic Acid). Its major role is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins. DNA polymerase is the builder enzyme that replicates DNA molecules in order to build a new strand of DNA. Ligase is the gluer. which helps glue DNA fragments together to form a new strand of DNA. Let us now proceed to the three major steps of DNA replication (initiation, elongation, and termination) and see what happens in each stage. Step 1: Initiation DNA replication starts at the Origin of Replication. The unzipping enzyme Helicase, ‘causes the DNA strand separation, which leads to the formation of the replication fork. It breaks the hydrogen bond between the base pairs to separate the strand thus separating the DNA into individual strands. Supercoiled DNA makes for efficient storage in the nucleus of a cell, but it poses problems during DNA replication, which occurs during cell division. During DNA replication, both the chromosomal supercoil and the double helix must be unwound to make room for the addition of a new strand to the double-stranded molecule. This adds pressure to the DNA that is still coiled and also has the potential to create a messy tangle of uncoiled DNA. Topoisomerase enzymes help prevent these situations during DNA replication by creating tiny cuts in the DNA strand to help it unravel the extra tension and make room for the replication machinery. The topoisomerase definition applies to several enzymes thal perform these functions during DNA replication Next, we have the Single-stranded DNA-binding protein (SSB) that binds to single-stranded regions of DNA. This binding serves a variely of functions - it prevents the strands from hardening too early during replication, it protects the single-stranded DNA from being broken down by nucleases during repair, and it removes the secondary structure of the strands so that other enzymes are able to access them and act effectively upon the strands. DNA primase is another enzyme that is important in DNA replication. It synthesizes a small RNA primer, which acts as a ‘kick-starter’ for DNA polymerase. This enzyme is ultimately responsible for the creation and expansion of new strands of DNA1 Aropresentative potion of OA ‘ich about Pe undge replication Step 2: Elongation During elongation, DNA Polymerase Ill makes the new DNA strand by reading the nucleotides on complementary bes the ox template strand and i, binding one nucleotide after the other to generate a whole new complementary strand. se Ithelps in proofreading and repairing the new strand. DNA Polymerase is able to identity and backtrack any mispaired nucleotides and correct them immediately. The bases attached to each strand then pair up with the three nucleotides found in the cytoplasm. If it finds an Adenine (A) on the template, it will only add a Thymine (T). If it finds a Guanine (G) on the template, it will only add a Cytosine (C). ‘One of the template strands is read in a 3' to 5" direction, therefore the new strand will be formed in a 5’ to 3' direction. This newly formed strand is referred to as the leading strand, Along the leading strand, DNA primase only needs to synthesize an RNA primer once, in the ate DNA polymerase. This is because DNA polymerase is able to extend the new DNA strand by reading the template 3° to 5°, synthesizing in a 5' to 3° direction as noted beginning, to it above. 3 Free mucintkies are attracrag thee “["% 2 Thetme strand of the DNA sepacate Tho yeragen ben berger the eases break A 4 Once the new nucleate have ned up, ey ae yanescogether by the eneyne NA patymerase rece 8 be af]However, the other template strand (the lagging strand) is antiparallel and is therefore read in a5’ to 3' direction. Continuous DNA synthesis, as in the leading strand, would need to be in the 3' to 5' direction, which is impossible as DNA polymerase cannot add bases to the 5’ ends. Instead, as the helix unwinds, RNA primers are added to the newly exposed bases on the lagging strand and DNA synthesis occurs in fragments, but stil in the 5' to 3" direction as before. ‘These fragments are known as Okazaki fragments. Step 3: Termination In the previous steps of DNA replication, at the Origin, a Primer helps the DNA Polymerase to initiate the process. As the strand 5 Fol atienuclooneswejonectofonnacanplerepajacaooeechan 18 6feated, the primer has to be using hia pelyemerase, dn his way, two identical strands of ONAare formed removed. This is when DNA Polymerase | comes into the nam mm picture to replace the RNA nucleotides from the Primer [4 with DNA nucleotides to make sure it is DNA all the ol way through. When DNA sea Polymerase Ill adds td nucleotides to the lagging strand and forms Okazaki a \ fragments, it leaves a gap or two between the fragments. These gaps are filled by the ‘enzyme ligase which makes sure that everything else is connected. DNAAigase DNA-Polymerase (Pola) Topoisomerase NA Polymerase (PoI6) Helicase! Single strand, Binding proteins Figure 3. DNA Replication ProcessThe Replication process is considered complete once all the Primers are removed and Ligase has filled in all the remaining gaps between the Okazaki Fragments. This process gives us two identical copies of the original DNA molecule. This whole replication process is happening in billions of cells in your body even at this very moment. The original DNA is called the template DNA, while the replicate DNA is called the complement DNA, both templates are IDENTICAL. Now, you might ask this question; “Why is it important for DNA TO UNDERGO REPLICATION?” Well, itis important for DNA present in the nucleus to be replicated so that every new cell receives the appropriate number of chromosomes, This process is necessary for cell repair and arowth and reproduction in living organisms. 1.2 TRANSCRIPTION OF DNA TO RNA RNA (Ribonucleic Acid), unlike the double-stranded DNA, is a nucleic acid polymer with a single strand. It is composed of the four nucleotides adenine, uracil (replaced thymine in DNA), guanine, and cytosine which are represented by their first letter A, U, G, C. (The only difference with DNA is the Uracil). RNA is the first intermediate in converting the information from the DNA into proteins which is important for proper cellular function. Below is a short summary of the difference between DNA and RNA. DNA RNA Contains the sugar deoxyribose ‘one more OH group than deoxyribose) Contains the sugar ribose (ribose has Double-stranded molecule Single-stranded molecule Stable under alkaline Not stable under alkaline conditions conditions Storing and transferring Acts as a messenger between DNA and genetic information ribosomes to make proteins. Uses the bases adenine, Uses adenine, uracil, cytosine, and thymine, cytosine, and guanine guaninecytosine 7 cytosine ‘SNucleobases Pa Guanine Guanine % Base pair Adenine, Adenine Uractt Thymine sugar-phosphate: Nucleobases Nucleobases of ‘ONA’ of RNA DNA Ribonucleic acid Deoxyribonucleic acid Figure 4, Structure of DNA and RNA. RNA falls into three major categories: Messenger RNA (mRNA), Transfer RNA (tRNA) and Ribosomal RNA (rRNA). mRNA copies the genetic code from the DNA into a form that can be read and used to make proteins. mRNA transmits genetic information from the nucleus to the cell's cytoplasm. rRNA is situated in the cytoplasm of a cell, where we can find the ribosomes. TRNA leads the translation of mRNA into proteins, tRNA transfers amino acids to the ribosome that matches to each three-nucleotide codon of rRNA. The amino acids then can be combined together and processed to make polypeptides and proteins. Transcription in protein synthesis is the process where RNA is made from the DNA by copying the base sequence of the double-stranded DNA into a piece of a single stranded nucleic This transcription process is catalyzed by the enzyme RNA Polymerase. Transcription of DNA to form RNA takes place in the cell's nucleus, This process uses DNA as a model to make an RNA (mRNA) molecule. During transcription, a strand of mRNA is made that corresponds to a strand of DNA. Just like DNA replication, transcription also occurs in three major steps: initiation, elongation and termination.BD rymine Figure 5. Transcription process in eukaryotic cells. Step 1: Initiation Initiation is the start of transcription. It transpires when the enzyme RNA polymerase binds to a specific region of a gene which is called the promoter with the help of proteins called ‘transcription factors’. Transcription factors are proteins that control the rate of transcription; they too bind to the promoter sequences with RNA polymerase. This signals the DNA double-strand to unwind and open so the RNA polymerase enzyme can “read” the bases found in one of the DNA strands. With the open strands, one is considered as the template strand (anti-sense strand) and this will be used to generate the mRNA. The other is called the non-template strand (sense strand). The other strand of DNA that is not transcribed is called the coding strand After reading the bases, the RNA polymerase enzyme is now ready to make a strand of mRNA with a ‘complementary sequence of bases. Stop 2: Elongation Elongation is the addition of nucleotides to the mRNA strand. RNA polymerase reads the ‘opened DNA strand and forms the mRNA molecule with the use of complementary base pairs There is a short time during this process when the newly formed RNA is bound to the opened DNA. During this process of elongation, an adenine (A) in the DNA binds to an uracil (U) in the RNA. RNA polymerase does not need a primer during this process. It simply initiates the mRNAsynthesis from the starting point and then moves downstream reading the anti-sense strand from 3° to 5' and generating the mRNA from the 5' to 3’ end as it goes. Unlike helicase enzyme in DNA replication, RNA polymerase zips DNA back up as it goes keeping only 1020 bases exposed one at a time. Step 3: Termination Termination is the last step of the transcription process. This happens when the RNA polymerase enzyme reaches a stop or termination sequence in the gene. When the stop sequence or stop codon is reached, the enzyme detaches from the gene. The mRNA strand is now produced and it detaches from DNA. It carries with it the information encoded in the gene. DNA Transcription (RNA Synthesis) By the end of transcription, the DNA segment is transcribed to form the mRNA molecule. The template strand shown below with the sequence: TACTAGAG-CATT transcribes to form the mRNA AUGAU-CUC-GUAA, Remember to take note of the transcription pattern: Thymine to Adenine, Adenine to Ura‘ Cytosine to Guanine, and Guanine to Cytosine, Uracil is being synthesized instead of Thymine ‘as compared during DNA replication 1.3 POST-TRANSCRIPTIONAL MODIFICATION When a eukaryotic gene is transcribed in the nucleus, the primary transcript (freshly made RNA molecule) isn't yet considered a messenger RNA. Instead, i's an “immature” molecule called a pre-mRNA. The pre-mRNA has to go through some modifications to become a mature mRNA molecule that can leave the nucleus and be translated, These include splicing, capping, and the addition of a poly-A tail, all of which can potentially be regulated — sped up, slowed down, or altered to result in a different product. pre-mRNAs must have their ends modified, by the addition of a 5" cap (at the beginning) which helps protect the mRNA from degradation. While addition of a 3° poly-A tail (at the end) signals the export of the cellular factors that the transcript needs to the cytoplasm Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions. These noncoding regions are called intervening sequences, or introns. The other regions are called exons because they are eventuallyexpressed, usually translated into amino acid sequences. RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence. In some cases, RNA splicing is carried out by spliceosomes. Spliceosomes consist of a variety of proteins and several small RNAs that recognize the splice sites. The RNAS of the spliceosome also catalyze the splicing reaction A Figure 7, Post-transcriotonal mocification of pre-mRNA By the end of transcription, mature mRNA has been made. This acts as the messaging system to allow translation and protein synthesis to ocour. 1.4 TRANSLATION OF RNA TO PROTEIN During translation, a polypeptide chain specified by the mRNA is synthesized. Each sequence of three nucleotide bases in the mRNA constitutes a codon, which specifies one amino acid in the polypeptide chain, or a start or stop signal. Translation requires RNAS and cell machinery, including ribosomes, Basic ingredients are the various types of RNAs produced in transcription and some proteins or enzymes. Protein Structure. Proteins may generally have globular or fibrous A structures depending on their particular role in the bodily functions. Globular proteins are spherical, compact, and soluble. Fibrous proteins are ‘elongated and insoluble, However, these two structure types may exhibit one or moretypes of protein structures. The protein building block is the amino acid. Amino acids combine through a dehydration link called a peptide bond. When several groups of amino acids are joined together, a protein macromolecule is formed. This is why proteins are considered as polymers of amino acids. Proteins are typically made of a chain of 20 amino acids. The human body makes any protein it needs by using a ‘combination of these 20 amino acids. Most amino acids have a structural template where an alpha carbon is bonded to the following forms: “A hydrogen atom (H) “A carboxyl group (COOH) “An amino group (-NH2) *A “variable” group The “variable” group is most responsible for difference as all of them have hydrogen, carboxyl group, and amino group bonds. Amino acids are linked through dehydration synthesis, peptide bonds are formed. Amino acids linked together by polypeptide bonds forms a polypeptide chain. When polypeptide chains are twisted, a 3-D shape forms a protein. Figure 9. A guide to 20 common amino acids. These amino acids are grouped as: essential and non-essential, Non-essential amino acids are those which the human body is capable of synthesizing, whereas essential amino acids must be obtained from the diet. Essential Symbol Non-Essential Amino Acids Symbol Amino Acidshistidine His alanine Ala isoleucine lle arginine Arg leucine Leu asparagine Asn sine Lys aspartic acid Asp methionine Met cysteine Cys phenylalanine Phe glutamic acid Glu threonine Thr glutamine Gin tryptophan Trp alycine Gly valine Val proline Pro serine Ser tyrosine Tyr Since the proteins formed by amino acids are incredibly huge and bulky molecules, it is very time-consuming and difficult to draw out their chemical structure in a similar way we draw smaller molecules. The common amino acids that make up proteins are given codes that represent them as shown in the table above. This makes describing the molecules so much easier. Proteins are synthesized in the human body through a process called translation. Translation occurs in the evtoplasm and involves converting genetic codes into proteins. Genetic codes are assembled during DNA transcription, where DNA is decoded into RNA. Cell structures called ribosomes then help transcribe RNA into polypeptide chains that need to be modified to become functioning proteins.A Figure 10. Translation of mRNA to form a polypeptide chain ofthe protein. The key components required for translation are mRNA, tRNA, ribosomes, and aminoacyl tRNA. synthetases, These four structures are briefly explained below: Ribosome The ribosome is a complex organelle, present in the cytoplasm, which serves as the site of action for protein synthesis. It provides the enzymes needed for peptide bond formation. The nucleotide sequence in mRNA is recognized in triplets, called codons. The ribosome moves along the single-strand mRNA, and when a complimentary codon sequence belonging to amino acid-bearing tRNA bonds with the mRNA, the amino acid is added to the chain. The mRNA possesses a stop codon, a sequence of three nucleotides that indicates that translation is complete. Upon reaching the stop codon, the ribosome ceases translation and releases the mRNA and newly generated polypeptide. Messenger RNA (mRNA) MRNA is used to convey information from DNA to the ribosome. It is a single strand molecule, complimentary to the DNA template, and is generated through transcription. Strands of mRNA. are made up of codons, each of which signifies a particular amino acid to be added to the polypeptide in a certain order, mRNA must interact with ribosomal RNA (rRNA), the central component of ribosomal machinery that recognizes the start and stop codons of mRNA, and transfer RNA (tRNA), which provides the amino acid once bound with a complimentary mRNA ‘codon. Transfer RNA (tRNA) This is a single strand of RNA composed of approximately 80 ribonucleotides. Each tRNA is read as a ribonucleotide triplet called an anticodon that is complementary to an mRNA codon. {RNA carry a particular amino acid, which is added to the growing polypeptide chain if ‘complimentary codons bond. ‘Aminoacyl tRNA synthetasesThese are enzymes that link each amino acid to its corresponding tRNA with the help of a two-step process. Each amino acid has a unique synthetase and the active site of each enzyme fits only one specific combination of the amino acid and tRNA. (14) There are three major steps in translation: initiation, elongation, and termination. These steps are briefly discussed below: Step 1: Initiation Initiation isthe first stage of translation. Proteins called initiation factors position the tRNA on the ribosomal surface at the P site (for peptidyl), where A peplide bonds will form, Nearby, two other sites wil form: the A site (for aminoacy)), where successive amino acid-bearing tRNAs will bind, and the E site (for exit), where empty tRNAs will exit the ribosome. Initiation factors bind to the small ribosomal subunit, which then binds to mRNA in the region of AUG, the start codon. Then the small subunit moves along the mRNA unti it reaches the start codon. Initiation factors bring in the large subunit that completes the translation initiation complex. Step 2: Elongation Elongation is a cyclic process in which amino acids are added one by one to the growing polypeptide chain. Elongation proceeds in the 5’ to 3° direction along the mRNA. Elongation ‘occurs in three steps: codon recognition, peptide bond formation, and translocation. Codon recognition involves the ribosome moving along the mRNA to the next codon. When a tRNA molecule with the appropriate anticodon appears, proteins called elongation factors assist in binding it to the exposed mRNA codon at the A site. When the second tRNA binds to the ribosome, it places its amino acid directly adjacent to the initial methionine, which is still attached to its tRNA molecule, which in tum is still bound to the ribosome. The two amino acids undergo a chemical reaction, catalyzed by peptidyl transferase, which releases the initial methionine from its RNA and attaches it instead by a peptide bond to the second amino acid. In this stage, a peptide bond is formed between the amino acid carried by the tRNA at the A site and the growing polypeptide chain attached to the tRNA at the P site. Following peptide bond formation, translocation takes place. The tRNA at the P site, which is now uncharged, is released from the ribosome's E site (exit site). The ribosome thenmoves one codon down the mRNA, shifting the {RNA carrying the growing polypeptide chain from the A site to the P site. This movement exposes the next codon on the mRNA for recognition, allowing for the continuation of translation Figure 12. Elongation Stage of Translation Step 3: Termination Termination, the final stage of translation, occurs when the ribosome reaches one of three stop codons (UAA, UAG, and UGA) for which there is no corresponding tRNA. The A site binds to a release factor, which triggers the release of the completed polypeptide chain and dissociation of the translation complex. The release factor causes the addition of a watermolecule instead of an amino acid. This reaction releases the polypeptide, and the translation assembly comes apart. The polypeptide chains produced during translation undergo some post-translational modifications, such as folding, before becoming a fully active protein. Figure 12. Termination Stage of Translation Below is a chart of all the mRNA codons and the amino acids they code for, Decoding codons is a task made simple because of the codon chart. Just start at the center of the chart for the first letter, Move to the outside next ring for the second letter and finally, find the final letter among the smallest set of letters in the third ring, Then you can read the amino acid in that sector. ‘To decode the codon for CAC, find the first letter C in the set of bases at the center of the circle, Then find the letter A in the second ring, then C in the third ring, There, you will read the amino acid in this sector as Histidine. Some of these codons are special. AUG is the start codon that initiates translation by coding for Methionine, And these three are stop codons: UAA, UAG and UGA, These are the ones that terminate translation.Figure 14. Genetic Code Wheel Chart In conclusion, the central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins. It outlines the fundamental processes of transcription, where DNA is transcribed into RNA, and translation, where RNA is translated into proteins. The central dogma is a fundamental principle that underlies the functioning of all living organisms. Proteins play a crucial role in various biological processes and are essential for the structure, function, and regulation of cells and organisms. They are involved in enzymatic reactions, signal transduction, cell communication, structural support, transportation of molecules, and immune responses, among many other functions. Proteins are the workhorses of cells, carrying out most of the cellular functions necessary for life. 2. ADVANCES AND DISCOVERIES IN CENTRAL DOGMA RESEARCH 1 Discovery of intronsUnlike bacterial genes, most eukaryotic genes are larger than they need to be to produce the polypeptides they code for. INTRONS are long sequences of nucleotides found in eukaryotic genes, which do not code for any portion of the polypeptide specified by the gene. Introns are inserted between EXONS, much shorter sequences in the gene that do code for portions of the polypeptide. In a typical human gene, the introns can be 10 to 30 times larger than the exons. In the late 1970s, the previously conceived notion that every nucleotide within a bacterial gene transcript is part of an amino acid-specifying codon was disproved. Biologists discovered that some aspects of prokaryotic gene expression did not apply to eukaryotes, as eukaryotic proteins are encoded by RNA segments that are cut from several locations along what is called the primary RNA transcript and then spliced together to form the mRNA that is eventually translated in the cytoplasm. When the researchers examined the resulting hybrid DNA molecules with an electron microscope, they found that the DNA appeared with some unpaired loops instead of a single duplex. They then concluded that nucleotide sequences must have been removed from the gene transcript before it appeared as cytoplasmic mRNA, These removed sequences are introns, and the remaining sequences are exons. Because introns are removed from the RNA transcript before it is translated into protein, they do not affect the structure of the protein encoded by the gene in which they occur, 2. RNA Splicing and Alternative Splicing RNA splicing is a process where introns are removed, and exons are joined together to produce mature mRNA. When a gene is transcribed, the primary RNA transcript contains sequences complementary to the entire gene, including introns as well as exons. However, in a process called RNA processing, or splicing, the intron sequences are cut out of the primary transcript before it is used in polypeptide synthesis and are not translated. The remaining sequences, the exons, are spliced together to form the final, “processed! mature” mRNA molecule that is translated. Alternative splicing allows a single gene to generate multiple mRNA transcripts, increasing protein diversity. Advances in RNA sequencing technologies have facilitated the discovery and characterization of alternative splicing events, shedding light on their functional implications.A 3. Differences between Bacterial and Eukaryotic Gene Expression A A Bacterial Gene Expression Eukaryotic Gene Expression Except for a few genes in the Most eukaryotic genes possess introns. Archaebacteria, prokaryotic genes lack introns. Individual bacterial MRNA molecules Eukaryotic mRNA molecules rarely often contain transcripts of several contain transcripts of more than one genes. gene. Bacteria often begin the translation of an Their mRNA molecules must be completely mRNA molecule before its transcription is formed and must pass across the nuclear completed. membrane before they are translated.Translation begins at an AUG codon mRNA molecules are modified at the 5” preceded by a special nucleotide leading end after transcription, adding a sequence. 5'cap, a methylated guanosine triphosphate. The cap initiates translation by binding the mRNA, usually at the first AUG, to the small ribosomal subunit. Eukaryotic mRNA molecules are modified before they are translated. The ribosomes of eukaryotes are a ttle larger than those of bacteria. 4, Non-Coding RNAS Formerly considered "junk DNA," they are found to be produced by non-coding regions of the genome. They play important roles in gene regulation and control gene ‘expression by targeting mRNA degradation or translational inhibition 5. Epigenetics Epigenetics is the study of the changes in the packaging and processing of identical DNA sequences due to external factors and internal cellular signals and how they can alter the expression of genes and traits without changes in the DNA nucleotide sequence. Epigenetic modifications, such as DNA methylation, histone modification, and chromatin remodeling, influence whether genes are turned “on” or “off.” Gene activation is blocked when DNA methyltransferases bind methyl molecules to DNA at sites where a phosphate links a cytosine and guanine. 6. Ribonucleoprotein Complexes Studies on ribonucleoprotein complexes, such as the spliceosome and the ribosome, have uncovered their intricate structures and functions, High-resolution structural techniques, including cryo-electron microscopy, have provided insights into the molecular mechanisms of RNA processing and translation 7, Transcriptional Regulation Researchers have discovered various mechanisms involved in transcriptional regulation, including transcription factors, enhancers, silencers, and chromatin looping. The development of techniques like ChiP-seq (chromatin immunoprecipitalion followedby sequencing) and genome editing tools (such as CRISPR-Cas9) has allowed for a more comprehensive understanding of gene regulatory networks. 8, mRNA Modifications Advances in RNA sequencing technologies have enabled the discovery and characterization of mRNA modifications, such as Né-methyladenosine (m6A) and 5-methylcytosine (m5C). These modifications can influence mRNA stability, translation efficiency, and protein synthesis. 3. APPLICATIONS IN LIVING THINGS, BIOTECHNOLOGY, AND. MEDICINE How a firefly's tail glows A firefly's tail glows due to a chemical reaction inside its abdomen. The produetion of ‘emission of light by a living organism is called bioluminescence. Fireflies contain luciferin, an organic compound that generates tight, and luciferase, an enzyme that produces bioluminescence. The oxygen reacts to luciferin, catalyzed with the help of luciferase and energy in the form of ATP (adenosine triphosphate). They go through a process called oxidation.The luciferin then becomes agitated and excited, elevating its energy level. When the excited luciferin drops back to its normal state, it releases that energy in the form of light. This process is possible because transcription ocours when the RNA polymerase copies the luciferase gene, which specifies the sequence of amino acids that make up the luciferase ‘enzyme in the form of messenger RNA. Fireflies are capable of generating light because of the many cells that are in its tail which produce thousands of luciferase enzymes at once. New ‘enzymes bind to a chemical called luciferin which then forms oxyluciferin Production of insulin in humans Insulin is a hormone that controls the human body's blood glucose levels and metabolism ~ the process that tums the food you eat into energy. Insulin allows cells in the muscles, liver, and fat to take up glucose and use it as a source of energy so they can function properly. Moreover, insulin regulates the body's energy supply by balancing micronutrient levels during the fed state. Insulin is mainly secreted by beta (B) cells in the islets of Langerhans of the pancreas, Inside the nucleus of the beta cells, the DNA containing the insulin gene is transcribed into a ‘complementary messenger RNA (mRNA) molecule. The mRNA molecule carries the genetic information from the insulin gene and moves out of the nucleus into the cytoplasm of the cell. It then binds to ribosomes in the cytoplasm, which are responsible for protein synthesis. tRNA molecules transport amino acids based on mRNA's genetic code. Ribosomes facilitate peptide bond formation between the amino acid and the growing protein chain. The ribosome reads theMRNA sequence and synthesizes a chain of amino acids according to the genetic code. This chain undergoes folding to form the insulin protein. Following synthesis, the insulin protein undergoes post-translational modifications that contribute to its maturation and functionality These modifications involve enzymatic cleavage and the addition of chemical groups. After modification, the insulin protein is directed to specific cellular compartments, particularly the secretory vesicles of beta cells. Within these specialized vesicles, the insulin molecules are stored until they are ready to be released into the bloodstream, CRISPR-Cas9 CRISPR-Cas9 is a technology that allows scientists to change a cell's DNA ata precise location. It is widely used in research and has great potential for therapy. Treatments using CRISPR-Cas9 technology are being developed for several genetic diseases, including sickle cell disease and cystic fibrosis. Scientists can use CRISPR-Cas9 to knock out a gene so that it is not expressed, or edit the gene to correct a disease-causing mutation. Gene therapy Gene therapy is an experimental technology that allows researchers to provide functioning copies of genes to cells with disease-causing versions of those genes. The therapeutic genes can be delivered inside cells in a variety of ways. One common delivery method uses viral vectors—modified viruses in which harmful portions of the virus genome are replaced with the therapeutic gene. When a viral vector is introduced into the body, it infects target cells and releases its genome, including the therapeutic gene, inside. The cell's machinery then produces a functional protein from the introduced gene. Gene Switches ‘One class of genetic technologies targets noncoding, regulatory DNA sequences in the genome, referred to as gene switches. These switches bind regulatory proteins that turn the transcription of genes on or off. Depending on the regulatory proteins present in a cell, genes can be expressed in different tissues, at different stages of development, or in response to certain stimuli. Scientists have begun to explore ways of interfering with gene switches to alter gene expression. Such methods can be used either to prevent genes linked to diseases from being turned on or to keep beneficial genes from being turned off. Exon SkippingExon skipping is a technology that changes how the primary RNA transcript of a gene with a disease-causing mutation is spliced, removing the mutation from the resulting mRNA. Scientists design a short segment of single-stranded RNA (called antisense RNA or antisense oligonucleotide) to bind to a sequence of the primary RNA transcript for a gene that is normally recognized by the cell's splicing machinery. When the antisense RNA is introduced in a patient's cells, it binds to the target sequence in the primary RNA transcript, causing the splicing machinery to “skip over" a segment of the transcript. Depending on how the antisense RNA is designed, one or more exons may be spliced out of the resulting mature mRNA, including the ‘exon with the disease-causing mutation. RNA Interference RNA interference (RNAi) technologies involve small RNA segments that target various. MRNAS for destruction, reducing the expression of certain genes. The RNAi method that's most well-studied for its potential in therapy uses small interfering RNA (siRNA). Scientists synthesize a short double-stranded RNA segment with a sequence complementary to that of a target ‘sequence and introduce it into the body. The double-stranded siRNA is taken up by cells, where it is recognized as being foreign, possibly from a virus, and cleaved into smaller pieces. Single RNA strands from the siRNA pieces are then incorporated into a cellular protein complex, called RNA-induced silencing complex (RISC), The siRNA guides the RISC to an mRNA with a ‘complementary sequence and cleaves it. By synthesizing different siRNAs, scientists can potentially silence any gene. Small Drug Molecule ‘Small molecule drugs, like aspirin, Gleevec, and ivacator, make up over 90 percent of the drugs on the market today. Small-molecule therapies comprise an incredibly diverse group of ‘chemical compounds of low molecular weight that are synthesized in the lab. Because of their small sizes, these molecules can easily be taken up by cells and may be administered to patients as pills or by injections. Small-molecule drugs may interact directly with disease-causing proteins, or through other molecules. Some small-molecule drugs block the negative effects of disease-causing proteins, whereas others restore their proper functioning. References: CK-12 Foundation. (n.d.). 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Overview of Transcription. Khan Academy. Retrieved from https:/www khanacademy org/science/ap-biology/gene-expression-and-regul ation/transcription-and-rna-processing/aloverview-of-transoription ‘Study.com. (n.d.). Topoisomerase: Overview & Function. Retrieved from https://study com/learn/lesson/topoisomerase-overview-function html What is the “Central Dogma’? (n.d.). @Yourgenome : Science Website. https:/www.yourgenome. org/facts/what-is-the-central-dogmal