Chapter 5 - Nucleic Acids PDF

CHAPTER 5 Nucleic Acids Image: https://www.helmholtz-hips.de/en/news-events/news/detail/news/mirror-mirror-new- enzyme-group-converts-amino-acid-into-its-mirror-image/ Types of nucleic acids (1) deoxyribonucleic acid (DNA) Nea...

CHAPTER 5 Nucleic Acids Image: https://www.helmholtz-hips.de/en/news-events/news/detail/news/mirror-mirror-new- enzyme-group-converts-amino-acid-into-its-mirror-image/ Types of nucleic acids (1) deoxyribonucleic acid (DNA) Nearly all the DNA is found within the cell nucleus Its primary function is the storage and transfer of genetic information. DNA is passed from existing cells to new cells during cell division. (2) ribonucleic acid (RNA) RNA occurs in all parts of a cell. It functions primarily in synthesis of proteins, the molecules that carry out essential cellular functions. The components of these include a (a) five-carbon (pentose) sugar, (b) phosphate, and (c) four heterocyclic amines called nitrogenous bases. Chemical composition of DNA and RNA Nucleotides: Structural Building Blocks for Nucleic Acids A nucleic acid is an unbranched polymer containing monomer units called nucleotides. A nucleotide is a three-subunit molecule in which a pentose sugar is bonded to both a phosphate group and a nitrogen-containing heterocyclic base. Nucleosides are produced by the combination of a sugar, either ribose (in RNA) or 2′-deoxyribose (in DNA), with a purine or a pyrimidine base. Nucleotides: (a) pentose sugar The sugar unit of a nucleotide is either the pentose ribose or the pentose 2′-deoxyribose. Structurally, the only difference between these two sugars occurs at carbon 2′. The —OH group present on this carbon in ribose becomes an —H atom in 2′-deoxyribose. (The prefix deoxy- means “without oxygen.”) Pentose unit in RNA Pentose unit in DNA Nucleotides: (b) nitrogen-containing heterocyclic bases Nucleotides: (b) nitrogen-containing heterocyclic bases Caffeine is a derivative of purine Thiamine (vit B) is a derivative of pyrimidine Nucleotides: (c) phosphate Phosphate, the third component of a nucleotide, is derived from phosphoric acid (H3PO4). Under cellular pH conditions, the phosphoric acid loses two of its hydrogen atoms to give a hydrogen phosphate ion (HPO42−). Nucleotides: Structural Building Blocks for Nucleic Acids -osine for purines or -idine for pyrimidines (Uracil to uridine) β-N-glycosidic bond Nucleotides: Structural Building Blocks for Nucleic Acids Nucleotide Formation The formation of a nucleotide from a sugar, a base, and a phosphate can be visualized as a two-step process. 1. First, the pentose sugar and nitrogen-containing base react to form a two-subunit entity called a nucleoside (not nucleotide, s versus t). 2. The nucleoside reacts with a phosphate group to form the three-subunit entity called a nucleotide. It is nucleotides that become the building blocks for nucleic acids. Nucleoside = sugar + base Nucleotide = nucleoside + phosphate Nucleotide Formation: (1) nucleoside formation Important characteristics of the process: (1) The base is always attached to C1′ of the sugar, which is always in a β-configuration. For purine bases, attachment is through N9; for pyrimidine bases, N1 is involved. The bond connecting the sugar and base is a β-N-glycosidic linkage Nucleotide Formation: (1) nucleoside formation Important characteristics of the process: (2) A molecule of water is formed as the two molecules bond together; a condensation reaction occurs. ▶ Nucleosides are more soluble in water than free heterocyclic bases because of the hydrophilic nature of the pentose’s -OH groups. Nucleotide Formation: (1) nucleoside formation Nucleosides are named as derivatives of the base that they contain; the base’s name is modified using a suffix. 1. For pyrimidine bases, the suffix -idine is used (cytidine, thymidine, uridine). 2. For purine bases, the suffix -osine is used (adenosine, guanosine). 3. The prefix deoxy- is used to indicate that the sugar present is deoxyribose. No prefix is used when the sugar present is ribose.  Nucleotide Formation Nucleotides are nucleosides that have a phosphate group bonded to the pentose sugar present. Important characteristics of the nucleotide formation process: 1. The phosphate group is attached to the sugar at the C5′ position through a phosphoester linkage. 2. As with nucleoside formation, a molecule of water is produced in nucleotide formation. Thus, overall, two molecules of water are produced in combining a sugar, base, and phosphate into a nucleotide. Nucleotide Formation Nucleotides are named by appending the term 5′-monophosphate to the name of the nucleoside from which they are derived.  the sequence in which nucleotides are Primary Structure of Nucleic Acids linked together in a nucleic acid.  Sugar-Phosphate Bases Backbone 5’ Because the sugar–phosphate backbone of a given nucleic acid does not vary, the primary structure of the nucleic acid depends only on the sequence of bases present. 5′ T–G–C–A 3′ 3’  Sugar-Phosphate Bases Backbone Important points about the 5’ nucleic acid primary structure: 1. Each nonterminal phosphate group of the sugar- phosphate backbone is bonded to two sugar molecules through a 3′,5′- phosphodiester linkage. There is a phosphoester bond to the 5′ carbon of one sugar unit and a phosphoester bond to the 3′ carbon of the other sugar. 3’  Sugar-Phosphate Bases Backbone Important points about the 5’ nucleic acid primary structure: 2. A nucleotide chain has directionality. One end of the nucleotide chain, the 5′ end, normally carries a free phosphate group attached to the 5′ carbon atom. The other end of the nucleotide chain, the 3′ end, normally has a free hydroxyl group attached to the 3′ carbon atom. By convention, the sequence of bases in a nucleic acid strand is read from the 5′ end to the 3′ end. 3’  Sugar-Phosphate Bases Backbone Important points about the 5’ nucleic acid primary structure: 3. Each nonterminal phosphate group in the backbone of a nucleic acid carries a 1– This behavior by the many charge. The parent phosphate groups in a phosphoric acid molecule nucleic acid backbone from which the phosphate gives nucleic acids their was derived originally had acidic properties. three –OH groups. Two of these become involved in the 3′,5′-phosphodiester linkage. The remaining –OH group is free to exhibit acidic behavior—that is, to produce an H+ ion. 3’ Three parallels between protein and nucleic acid primary structure: 1. DNAs, RNAs, and proteins all have backbones that do not vary in structure. Three parallels between protein and nucleic acid primary structure: 2. The sequence of attachments to the backbones (nitrogen bases in nucleic acids and amino acid R groups in proteins) distinguishes one DNA from another, one RNA from another, and one protein from another. Three parallels between protein and nucleic acid primary structure: 3. Both nucleic acid polymer chains and protein polymer chains have directionality; for nucleic acids, there is a 5′ end and a 3′ end, and for proteins, there is an N-terminal end and a C- terminal end. DNA structure: the double helix Like proteins, nucleic acids have secondary, or three-dimensional, structure as well as primary structure. The secondary structures of DNAs and RNAs differ, so they will be discussed separately. The amounts of the bases A, T, G, and C present in DNA molecules were the key to determination of the general three-dimensional structure of DNA molecules. %A = %T %C = %G %purines (A,G) = %pyrimidines (C,T) In 1953, an explanation for the base composition patterns associated with DNA molecules was proposed by the American microbiologist James Watson and the English biophysicist Francis Crick. Their model, which has now been validated in numerous ways, involves a double-helix structure that accounts for the equality of bases present, as well as for other known DNA structural data. The X-ray diffraction studies of Rosalind Franklin and Maurice Wilkens revealed several repeat distances that characterize the structure of DNA: 0.34 nanometers (nm), 3.4 nm, and 2 nm. With this information, Watson and Crick concluded that DNA is a double helix of two strands of DNA wound around one another. The structure of the double helix is often compared to a spiral staircase. The sugar-phosphate backbones of the two strands of DNA spiral around the outside of the helix like the handrails on a spiral staircase. The nitrogenous bases extend into the center at right angles to the axis of the helix. You can imagine the nitrogenous bases forming the steps of the staircase. Key features: 1. Two strands of DNA form a right-handed double helix. 2. The bases in opposite strands hydrogen bond according to the AT/GC rule. Key features: 3. The two strands are antiparallel with regard to their 5’ to 3’ directionality (One strand runs in the 5′-to-3′ direction, and the other is oriented in the 3′-to-5′ direction.) 4. There are ~10.0 nucleotides in each strand per complete 360° turn of the helix. Image: https://apbiologyctd.wordpress.com/genetics/dna-replication/ DNA structure: the double helix (base pairing) Why do only A-T and C-G base pairings occur? A physical restriction, the size of the interior of the DNA double helix, limits the base pairs that can hydrogen-bond to one another. Only pairs involving one small base (a pyrimidine) and one large base (a purine) correctly “fit” within the helix interior. There is not enough room for two large purine bases to fit opposite each other (they overlap), and two small pyrimidine bases are too far apart to hydrogen-bond to one another effectively. In addition, hydrogen- bonding possibilities are most favorable for the A–T and G–C pairings with 2 and 3 H-bonds, respectively. DNA structure: the double helix Hydrogen bonding interactions Hydrogen bonding between base pairs is an important factor in stabilizing the DNA double- helix structure. Although hydrogen bonds are relatively weak forces, each DNA molecule has so many base pairs that, collectively, these hydrogen bonds are a force of significant strength. In addition to hydrogen bonding, base-stacking interactions contribute to DNA double-helix stabilization. Base-stacking interactions The bases in a DNA double helix are positioned with the planes of their rings parallel (like a stack of coins). Stacking interactions involving a given base and the parallel bases directly above and below it also contribute to the stabilization of the DNA double helix. Purine and pyrimidine bases are hydrophobic in nature. (Hydrophobic interactions involving the nonpolar tails of membrane lipids contribute to the structural stability of cell membranes (Section 8-10), and hydrophobic interactions involving nonpolar R groups of amino acids contribute to protein tertiary structure stability. Image: https://pubs.acs.org/doi/10.1021/acs.jctc.8b00643 DNA structure: chromosomes Chromosomes are pieces of DNA that carry the genetic instructions, or genes, of an organism. Organisms such as the prokaryotes (no true nucleus surrounded by a nuclear membrane and there are no true membrane-bound organelles) have only a single chromosome, and its structure is relatively simple. Others, the eukaryotes (have cells containing a true nucleus enclosed by a nuclear membrane), have many chromosomes, each of which has many different levels of structure. The complete set of genetic information in all the chromosomes of an organism is called white beads: circular DNA molecule the genome. blue and red beads: non-associating proteins (red beads denote the DNA-binding sites of each protein chain). Yellow balls: macromolecular “crowders”; mainly the Image: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1116776/full non-binding globular proteins and RNA. DNA structure: chromosomes The number and size of the chromosomes of eukaryotes vary from one species to the next. For instance, humans have twenty-three pairs of chromosomes, while the adder’s tongue fern has 631 pairs of chromosomes. But the chromosome structure is the same for all those organisms that have been studied. The first level of structure is the nucleosome, which consists of a strand of DNA wrapped around a small disk made up of histone proteins. The nucleosomes then coil into a larger structure called the condensed fiber. This complex of DNA and protein is termed chromatin and makes up the eukaryotic chromosomes. DNA structure: chromosomes A histone is a protein that provides structural support for a chromosome. Each chromosome contains a long molecule of DNA, which must fit into the cell nucleus. To do that, the DNA wraps around complexes of histone proteins, giving the chromosome a more compact shape. The full complexities of the eukaryotic chromosome are not yet understood, but there are probably many such levels of coiled structures.  A karyotype is an individual’s complete set of chromosomes. The term also refers to a laboratory- produced image of a person’s chromosomes isolated from an individual cell and arranged in numerical order. A karyotype may be used to look for abnormalities in chromosome number or structure. https://www.genome.gov/genetics-glossary/Karyotype  Some human genetic disorders are characterized by unusual chromosome numbers. Disorder Characterized by Down syndrome an extra copy of chromosome 21 Some traits: varying degrees of mental challenges, a flattened face, and short stature Edward syndrome An extra copy of chromosome 18 Some traits: extreme mental and physical defects and early death Patau syndrome An extra copy of chromosome 13 Some traits: extreme mental and physical defects and early death Klinefelter syndrome Males with two X chromosomes and one Y Some traits: show sexual immaturity and breast development XYY syndrome Males with an extra Y chromosome Some traits: unusually tall Triple X syndrome Females with an extra X chromosome Some traits: unusually tall, have problems with spoken language and processing spoken words, coordination problems, and weaker muscles Turner syndrome Females with only a single X chromosome Some traits: short stature, a webbed neck, and sexual immaturity It is thought that 50% of all miscarriages are the result of abnormal chromosome numbers. DNA replication DNA replication is the biochemical process by which DNA molecules produce exact duplicates of themselves. A cell that is missing a critical gene will die, just as an individual with a genetic disorder, a defect in an important gene, may die early in life. Thus, it is essential that the process of DNA replication produces an absolutely accurate copy of the original genetic information. If mistakes are made in critical genes, the result may be lethal mutations.  DNA replication Watson and Crick: Since the base- pair rule exists, Watson and Crick first suggested that an enzyme could “read” the nitrogenous bases on one strand of a DNA molecule and add complementary bases to a strand of DNA being synthesized. The product of this mechanism would be a new DNA molecule in which one strand is the original, or parent, strand and the second strand is a newly synthesized, or daughter, strand. This mode of DNA replication is called semiconservative replication.  DNA replication Matthew Meselson and Franklin Stahl (1958): E. coli cells were grown in a medium in which 15NH4+ was the sole nitrogen source. 15N is a nonradioactive, heavy isotope of nitrogen. Thus, growing the cells in this medium resulted in all of the cellular DNA containing this heavy isotope. The cells containing only 15NH4+ were then added to a medium containing only the abundant isotope of nitrogen, 14NH4+, and were allowed to grow for one cycle of cell division. When the daughter DNA molecules were isolated and analyzed, it was found that each was made up of one strand of “heavy” DNA, the parental strand, and one strand of “light” DNA, the new daughter strand. DNA replication: Overview 1. Chromatin disassembly (eukaryotes): ATP-dependent chromatin remodeling complexes facilitate the sliding or removal of nucleosomes from DNA ahead of the replication fork (10.1101/cshperspect.a010207) 2. DNA double helix unwinding: DNA helicase binds and breaks and the hydrogen bonds between complementary bases. The point at which the DNA double helix is unwinding, which is constantly changing (moving), is called the replication fork. 3. Primer binding: the enzyme primase generates short strands of RNA that bind to the single-stranded DNA to initiate DNA synthesis (one primer in the leading strand and multiple primers in the lagging strand) 4. Elongation: Polymerase binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand by forming new phosphodiester linkages. leading strand: continuous 5’ to 3’ direction (towards helicase) lagging strand: in short Okazaki fragments (elongation is away from the helicase) DNA replication: Overview 5. Termination: Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands, which are replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. DNA ligase joins Okazaki fragments together forming a single unified strand. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences (repeated DNA sequences that act as protective caps) at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape.  3’ 5’ DNA helicase breaks the hydrogen bonds between the DNA strands. This, in turn, causes supercoiling of the molecule  3’ 5’ Topoisomerase alleviates positive supercoiling ahead of the replication fork.  3’ Single-strand binding proteins 5’ keep the parental strands apart. They also protect the strands from degradation and prevent secondary structure formation.  3’ DNA polymerase III “reads” each 5’ parental strand (or template) and catalyzes the polymerization of a complementary daughter strand. Deoxyribonucleotide triphosphate (dNTP) molecules are the precursors for DNA replication. In this reaction, a pyrophosphate group is released as a phosphoester bond is formed between the 5′-phosphoryl group of the nucleotide being added to the chain and the 3′-OH of the nucleotide on the daughter strand. This is called 5′ to 3′ synthesis. DNA polymerase III “reads” each parental strand (or template) and catalyzes the polymerization of a complementary daughter strand.  ! 3’ Complicating factors: DNA pol III can only catalyze DNA chain elongation in the 5′ to 3′ direction but the two strands of DNA are antiparallel the need for an RNA 5’ primer to serve as the starting point for DNA replication  1 primer 3’ Multiple primers In the next step, primase catalyzes the 5’ synthesis of a small piece of RNA (10- 12 nucleotides) called an RNA primer that serves to “prime” the process of DNA replication.  1 primer 3’ For the leading strand, a single RNA Multiple primers primer is produced at the replication 5’ origin and DNA polymerase III continuously catalyzes the addition of nucleotides in the 5′ to 3′ direction, beginning with addition of the first nucleotide to the RNA primer.  1 primer 3’ On the lagging strand, many RNA Multiple primers primers are produced as the replication 5’ fork proceeds along the molecule. DNA pol III catalyzes DNA chain elongation from each of these primers. When the new strand “bumps” into a previous one, synthesis stops at that site.  1 primer Meanwhile, at the 3’ replication fork, a new primer is being synthesized by primase. On the lagging strand, many RNA Multiple primers primers are produced as the replication 5’ fork proceeds along the molecule. DNA pol III catalyzes DNA chain elongation from each of these primers. When the new strand “bumps” into a previous one, synthesis stops at that site.  1 primer The final steps of 3’ synthesis on the lagging strand involve (1) removal of the primers, (2) repair of the gaps, and (3) sealing of the fragments into an intact strand of DNA. Multiple primers 5’ The final steps of 3’ synthesis on the lagging strand involve (1) removal of the primers, (2) repair of the gaps, and (3) sealing of the fragments into an intact strand of DNA. DNA polymerase I (which is also an exonuclease) 5’ catalyzes the removal of the RNA primer and its replacement with DNA nucleotides. The final steps of 3’ synthesis on the lagging strand involve (1) removal of the primers, (2) repair of the gaps, and (3) sealing of the fragments into an intact strand of DNA. DNA ligase covalently links the DNA 5’ fragments together.  3’ DNA poly III is also able to proofread the newly synthesized strand. If the 5’ wrong nucleotide has been added to the growing DNA strand, it is removed and replaced with the correct one. In this way, a faithful copy of the parental DNA is ensured. (1) Initiation (2) Elongation (leading strand) (2) Elongation (lagging strand) (2) Elongation (lagging strand) (3) Termination The replisome complex dismantles after the convergence of the two replication forks (not shown) (In eukaryotes) Ribonuclease H (RNAse H) removes the RNA primer at the beginning of each Okazaki fragment, and DNA ligase joins these fragments together to create one complete strand. Telomeres Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes After the first round, the new lagging strand is shorter than its template. After the second round, both the leading and lagging strands have become shorter than the original parental DNA. Telomeres The ends of the linear chromosomes have specialized DNA “caps” known as telomeres: repetitive sequences that code for no particular gene. These telomeres protect the important genes from being deleted as cells divide and as DNA strands shorten during replication. In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times. However, even these sequences are not unlimited. After certain number of DNA replications, and hence cell divisions, the telomeres are shortened to an extent that it leads to replicative cell senescence or apoptosis.  Telomeres need to be protected from a cell's DNA repair systems because they have single-stranded overhangs, which "look like" damaged Telomeres DNA. In some species (including humans), the single-stranded overhangs bind to complementary repeats in the nearby double-stranded DNA, causing the telomere ends to form protective loops. Shelterin (also called telosome) is a protein complex known to protect telomeres in many eukaryotes from DNA repair mechanisms, as well as to regulate telomerase activity Chromosomes Once the DNA within a cell has been replicated, it interacts with specific proteins in the cell called histones to form structural units that provide the most stable arrangement for the long DNA molecules. These histone–DNA complexes are called chromosomes. A chromosome is an individual DNA molecule bound to a group of proteins. Typically, a chromosome is about 15% by mass DNA and 85% by mass protein. Chromosomes occur in matched (homologous) pairs. Homologous chromosomes have similar, but not identical, DNA base sequences; both code for the same traits but for different forms of the trait (for example, blue eyes versus brown eyes). Bacterial DNA Replication DNA replication begins at a unique sequence known as the replication origin. Replication occurs bidirectionally at the rate of about 500 new nucleotides every second! Bacterial DNA Replication The point at which the new deoxyribonucleotide is added to the growing daughter strand is called the replication fork. Since DNA synthesis occurs bidirectionally, there are two replication forks moving in opposite directions. Bacterial DNA Replication The point at which the new deoxyribonucleotide is added to the growing daughter strand is called the replication fork. Since DNA synthesis occurs bidirectionally, there are two replication forks moving in opposite directions. Eukaryotic DNA Replication DNA replication in eukaryotes is more complex. The human genome consists of approximately three billion nucleotide pairs. Just one chromosome may be nearly 100 times longer than a bacterial chromosome. To accomplish this huge job, DNA replication begins at many replication origins and proceeds bidirectionally along each chromosome. Eukaryotic DNA Replication DNA replication begins at many replication origins “bubbles” of newly synthesized DNA proceeds bidirectionally along each chromosome. https://apbiologyctd.wordpress.com/genetics/dna-replication/ at a glance DNA Replication Antimetabolites: Anticancer Drugs That Inhibit DNA Synthesis Cancer is a disease characterized by rapid uncontrolled cell division. Rapid cell division necessitates the synthesis of large amounts of DNA, as DNA must be present in each new cell produced. Antimetabolites are a class of anticancer drugs that interfere with DNA replication because their structures are similar to molecules required for normal DNA replication. Synthesis of adenine-containing nucleotides is inhibited when 6-MP is present; nonfunctional DNA results when 6-MP, rather than adenine, is incorporated into a nucleotide. Antimetabolites: Anticancer Drugs That Inhibit DNA Synthesis  Antimetabolites: Anticancer Drugs That Inhibit DNA Synthesis Folic acid, or vitamin B, is an essential and necessary element for the synthesis of nucleotides and other biomolecules after reduction by dihydrofolate reductase. Information Flow in Biological Systems The central dogma of the genetic info in the linear molecular biology states sequence of that in cells the flow of nucleotides is genetic information being translated contained in DNA is a one- into a protein, a way street that leads from linear sequence DNA to RNA to protein. of amino acids a single strand of DNA serves as a template for the synthesis of an RNA molecule Image: https://www.sciencefacts.net/central-dogma.html http://naturejournals.org/index.php/animalbio/ab- modules/technologies/protein-synthesis/ Information Flow in Biological Systems unlike DNA, does not contain equal amounts of specific bases. 5. Longer (50 million to 300 million 5. Shorter (75 nucleotides to a base pairs in humans) few thousand nucleotides) Image: https://howbiotech.com/dna-vs-rna-venn-diagram/  Types of RNA (1) Heterogeneous nuclear RNA (hnRNA) is RNA formed directly by DNA transcription. Post-transcription processing converts the heterogeneous nuclear RNA to messenger RNA. (2) Messenger RNA (mRNA) is RNA that carries instructions for protein synthesis (4) Ribosomal RNA (rRNA) is RNA that combines with (genetic information) to the sites for protein specific proteins to form ribosomes, the physical sites synthesis. The molecular mass of for protein synthesis. Ribosomes have molecular messenger RNA varies with the length of masses on the order of 3 million amu. The rRNA the protein whose synthesis it will direct. present in ribosomes has no informational function. (3) Small nuclear RNA (snRNA) is RNA that facilitates the conversion of hnRNA to (5) Transfer RNA (tRNA) is RNA that delivers amino mRNA. It contains from 100 to 200 acids to the sites for protein synthesis. Transfer RNAs nucleotides. are the smallest of the RNAs, possessing only 75–90 nucleotide units. Image: https://www.sciencefacts.net/central-dogma.html  https://en.wikipedia.org/wiki/Exon An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. In RNA splicing, introns are removed and exons are covalently joined to one another as part of generating the mature RNA. Just as the entire set of genes for a species constitutes the genome, the entire set of exons constitutes the exome. Information Flow in Biological Systems Transcription is the process by which DNA directs the synthesis of hnRNA/mRNA molecules that carry the coded information needed for protein synthesis. Messenger RNA production via transcription is actually a “two-step” process in which an (1) hnRNA molecule is initially produced and then (2) is “edited” to yield the desired mRNA molecule. The mRNA molecule so produced then functions as the carrier of the information needed to direct protein synthesis. Transcription: RNA synthesis Initiation Elongation/synth esis of the RNA transcript Termination Transcription: RNA synthesis The mechanics of transcription are in many ways similar to those of DNA replication. 1. Initiation: The first, called initiation, involves binding of RNA polymerase to a specific nucleotide sequence, the promoter, at the beginning of a gene. This interaction of RNA polymerase with specific promoter DNA sequences allows RNA polymerase to recognize the start point for transcription. It also determines which DNA strand will be transcribed. Unlike DNA replication, transcription produces a complementary copy of only one of the two strands of DNA. As it binds to the DNA, RNA polymerase separates the two strands of DNA so that it can “read” the base sequence of the DNA. Transcription: RNA synthesis Initiation Elongation/synth esis of the RNA transcript Termination Transcription: RNA synthesis 2. Chain elongation: chain elongation, begins as the RNA polymerase “reads” the DNA template strand and catalyzes the polymerization of a complementary RNA copy. One free ribonucleotide at a time align along one of the exposed strands of DNA bases, the template strand, forming new base pairs. Only about 10 base pairs of the DNA template strand are exposed at a time, U rather than T aligns with A in the base-pairing process. ribose, rather than deoxyribose, becomes incorporated into the new nucleic acid backbone RNA polymerase is involved in the linkage of ribonucleotides, one by one, to the growing hnRNA molecule. https://en.wikipedia.org/wiki/Coding_strand https://www.khanacademy.org/sc ience/biology/gene-expression- central-dogma/transcription-of- dna-into-rna/a/stages-of- transcription#:~:text=Transcriptio n%20termination,DNA%20known %20as%20a%20terminator. Transcription: RNA synthesis 3. Termination: The RNA polymerase finds a termination sequence at the end of the gene. The newly formed hnRNA molecule and the RNA polymerase enzyme are released, and the DNA then rewinds to re-form the original double helix.  Post-transcriptional Processing of RNA In eukaryotes, transcription produces a primary transcript that must undergo extensive post-transcriptional modification before it is exported out of the nucleus for translation in the cytoplasm. Eukaryotic primary transcripts undergo three (3) post- transcriptional modifications: (1) 5′-methylated cap structure The cap structure is required for efficient translation of the final mature mRNA. Post-transcriptional Processing of RNA (2) poly(A) tail The poly(A) tail protects the 3′ end of the mRNA from enzymatic degradation and thus prolongs the lifetime of the mRNA. https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/72-transcription-and-gene/messenger-rna.html 1200 nucleotides long, but only 438 nucleotides carry the genetic information for protein (3) RNA splicing (eukaryotes) the removal of portions of the primary transcript that are not protein coding there are “signals” in the DNA to mark the boundaries of the introns. Note: Bacterial genes are continuous; all the nucleotide sequences of the gene are found in the mRNA.  Recognition of the splice boundaries and stabilization of the splicing complex requires the assistance of particles called spliceosomes. (3) RNA splicing Spliceosomes are composed of a variety of small nuclear ribonucleoproteins (eukaryotes) (snRNPs, read “snurps”) -- consists of a small RNA and associated proteins. The RNA components of different snRNPs are complementary to different sequences involved in splicing. By hydrogen bonding to a splice boundary or intron sequences, the snRNPs recognize and bring together the sequences involved in the splicing reactions. In fact, it is the small RNA molecules that catalyze the splicing reactions. Such catalytic RNAs are called ribozymes. Alternative splicing Research now shows that the information-bearing sections of DNA within a gene can be spliced together an average of eight different ways. There could turn out to be around 150,000–200,000 relevant mRNA molecules as compared to 20,000–25,000 genes (genome-phenome gap) within the human genome. Alternative splicing is a process by which several different proteins that are variations of a basic structural motif can be produced from a single gene. In alternative splicing, an hnRNA molecule with multiple exons present is spliced in several different ways. Alternative splicing patterns that can occur when an hnRNA contains four exons, two of which are alternative exons. The “machinery” that bridges the genome–proteome “gap” is spliceosomes. It is spliceosomes that give humans their chemical complexity. Alternative splicing Exon B is Exon C is retained retained Both B and C Both B and C are retained are removed The Genetic Code The mRNA carries the genetic code for a protein. But what is the nature of this code? In 1954, George Gamow proposed that because there are only four “letters” in the DNA alphabet (A, T, G, and C) and because there are twenty amino acids, the genetic code must contain words made of at least three letters taken from the four letters in he DNA alphabet. How did he come to this conclusion? 4n two-letter words constructed from any combination of the four letters has a “vocabulary” of only (42) = 16 words (or combinations). In other words, there are only sixteen different ways to put A, T, C, and G together two bases at a time (AA, AT, AC, AG, TT, TA, etc.). That is not enough to encode all twenty amino acids. A code of four-letter words gives (44) = 256 combinations, far more than are needed. A code of three-letter words, however, has a possible vocabulary of (43) = 64 combinations, sufficient to encode the twenty amino acids but not too excessive. The Genetic Code is a sequence of amino acids in a mRNA that determines the amino acid order for the protein. A given triplet in mRNA contains a base sequence, transcribed from DNA, that translates to a specific amino acid. This triplet is called a codon. assigns all 20 amino acids to codons of mRNA. There are 64 possible codon combinations from the four bases A, G, C, and U. contains certain codons that signal the “start” and “end” of a polypeptide chain. The Genetic Code The three codons UGA, UAA, and UAG are stop signals for protein synthesis. The triplet AUG serves two purposes in protein synthesis. 1. It represents the start codon initiating protein synthesis if it is at the 5' end of an mRNA. 2. It codes for the amino acid methionine if it is found elsewhere in mRNA. Translation: Protein synthesis The process of protein synthesis is called translation. It involves translating the genetic information from the sequence of nucleotides into the sequence of amino acids in the primary structure of a protein. Translation: Protein synthesis Translation is carried out on ribosomes, which are complexes of ribosomal RNA (rRNA) and proteins. Each ribosome is made up of two subunits: a small and a large ribosomal subunit. In eukaryotic cells, the small ribosomal subunit contains one rRNA molecule and thirty-three different ribosomal proteins, and the large subunit contains three rRNA molecules and about forty-nine different proteins. Role of the transfer RNA (tRNA) The codons of mRNA must be read if the genetic message is to be translated into protein. The molecule that decodes the information in the mRNA molecule into the primary structure of a protein is transfer RNA (tRNA). To decode the genetic message into the primary sequence of a protein, the tRNA must faithfully perform two functions. 1. the tRNA must covalently bind one, and only one, specific amino acid 2. the tRNA must be able to recognize the appropriate codon on the mRNA that calls for that amino acid. 1. the tRNA must covalently bind one, and only one, specific amino acid 2. the tRNA must be able to recognize the appropriate codon on the mRNA that calls for that amino acid. Briefly, protein synthesis involves: Transcription, during which a complementary copy of DNA, called mRNA, is created by RNA polymerase. mRNA travels out of the nucleus to the ribosome. tRNA activation, during which a tRNA synthetase attaches the correct amino acid to the acceptor stem of the tRNA. Translation: Protein synthesis starts with the start codon. Activated tRNA, with methionine attached, enters the ribosome and hydrogen bonds to mRNA. The second activated tRNA, matching the next codon on mRNA, enters. The two amino acids join. The first tRNA leaves and the tRNA with the dipeptide attached shifts into the first position, a shifting called translocation. This process continues over and over until a protein chain emerges. Briefly, protein synthesis involves: (cont.) Termination Eventually the ribosome encounters a stop codon, and protein synthesis stops. there is no tRNA with an anticodon for the “stop” codons. The protein chain is released. The initial amino acid methionine is often removed from the beginning of the chain The protein chain folds into its tertiary structure that makes it a biologically active protein. Efficiency of mRNA Utilization