Molecular Biology Lecture Notes PDF

The structure of DNA, organisation of the genome, and the structure and types of RNA Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2024/2025 Structure of the nucleus  Nuclear envelope The nuclear envelope is made up of:...

The structure of DNA, organisation of the genome, and the structure and types of RNA Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2024/2025 Structure of the nucleus  Nuclear envelope The nuclear envelope is made up of: ❑ the outer (cytoplasmic) nuclear membrane ❑ the inner nuclear membrane ❑ the perinuclear space, which is located between the two membranes ❑ the nuclear lamina, which adheres to the inner nuclear membrane ❑ nuclear pores, which allow exchange of molecules with the cytoplasm Nuclear envelope  Nuclear lamina The nuclear lamina consists of a network of protein filaments ([nuclear] lamins) The structure and amino acid composition of lamins are similar to that of intermediate filaments The nuclear lamina: ❑ gives shape to the cell nucleus ❑ participates in the structural organisation of chromatin by providing a site of attachment for chromatin domains (lamina-associated domains (LADs)) Lamins are also involved in the process of fragmentation and reconstruction of the nuclear envelope during mitosis  Nuclear pores A nuclear pore consists of three rings, each of which are made of eight elements to form: ❑ the cytoplasmic ring - the outer nuclear membrane ❑ the central pore ❑ the nuclear ring - the inner nuclear membrane There are eight filaments anchored in both rings, respectively The nuclear filaments form a structure called the nuclear basket Nuclear pores  Nuclear pores Nuclear pores allow two-way and selective transport of macromolecules: ❑ from the nucleus to the cytoplasm ❑ from the cytoplasm to the nucleus  The nuclear matrix The nuclear matrix is a network of fibers located inside the nucleus It is involved in: ❑ regulation of DNA replication ❑ regulation of gene expression ❑ transcription and maturation of pre-RNA ❑ transport of ribosome precursors into the cytoplasm  The nucleolus The nucleolus during interphase occupies 25% of the volume of the nucleus The nucleolus disappears in late prophase and is reconstructed in telophase The main components are RNA and proteins  Nuclear chromatin Nuclear chromatin is the form of chromosomes during interphase The chemical composition of chromatin: ❑ deoxyribonucleic acid (DNA) - 36.5% ❑ histone proteins (basic) - 37.5% ❑ non-histone proteins (acidic) - 10.5% ❑ ribonucleic acid (RNA) - 9.5% ❑ water, calcium and magnesium ions  Nuclear chromatin DNA molecules in human cell are 2 m long (5.3 x109 base pairs), respectively In metaphase, the DNA molecule is made 10,000 times shorter (i.e., it occurs in a packed form) making the chromosome The packing of DNA is a result of: ❑ a specific structural organisation of the DNA in space ❑ the participation of histone and non-histone proteins  Nuclear chromatin Interphase chromosomes occupy a distinct space within the nucleus  Chromatin types When the cell is in interphase, chromatin in the nucleus is classified as: ❑ euchromatin ❑ completely or partially relaxed ❑ stains bright ❑ genetically active ❑ heterochromatin ❑ condensed ❑ stains dark ❑ genetically inactive  Euchromatin Euchromatin is formed by the transformation of chromosomes in metaphase into chromosomes in interphase Consists of fully developed or a lightly packed chromosomes (packing is ~ 1,000x) In interphase, euchromatin accounts for approximately 92% of the genome It is genetically active and contains DNA that is transcribed Due to the condensation of DNA strands, it can transform into packed (inactive) chromatin  Heterochromatin In interphase, the nucleus contains heterochromatin which: ❑ is DNA that is always in a condensed state ❑ is within the confines of the nuclear envelope Heterochromatin decondenses only before DNA replication in the late S phase Heterochromatin is located: ❑ at the ends of chromosomes (telomeric) ❑ within the centromere (centromeric) ❑ in some parts of the chromosomes (intercalary)  Heterochromatin Heterochromatin is divided into: ❑ constitutive, which occurs at the centromeres and telomeres of chromosomes ❑ facultative, which is formed by the reversible condensation of euchromatin that contains genes used when necessary Structure of chromatin  Nucleic acids Nucleic acids are made up of three types of compounds: ❑ heterocyclic nitrogenous bases ❑ a five-carbon sugar ❑ a phosphate group - a phosphoric acid residue The base-sugar unit is called a deoxyribonucleoside (DNA) or nucleoside A base-sugar-phosphate unit is called a deoxyribonuclotide (DNA) or nucleotide A [deoxyribo]nucleotide is the fundamental monomer unit of a nucleic acid chain  Nitrogenous bases Nitrogenous bases that build nucleic acids: ❑ are heterocyclic, aromatic compounds containing carbon and nitrogen atoms within the ring structures ❑ are derivatives of pyrimidine or purine, respectively Pyrimidine is a heterocyclic compound containing four carbon atoms and two nitrogen atoms in the 1 and 3 carbon positions Purine is a compound consisting of a pyrimidine ring connected to an imidazole ring Nitrogenous bases Pyrimidines Purines  Nitrogenous bases Pyrimidine bases (single-ring) : ❑ cytosine (C) ❑ thymine (T) ❑ uracil (U) Purine bases (two-ring): ❑ adenine (A) ❑ guanine (G)  The pentose sugars In RNA nucleotides, the sugar component is a pentose called ribose In DNA deoxyribonucleotides, the sugar component is a pentose called deoxyribose Both pentoses have: ❑ an oxygen within the ring ❑ the 5' carbon is outside of the ring Deoxyribose is devoid of a hydroxyl group at the 2' carbon. The pentose sugars D-ribose D-2-deoxyribose  Phosphate group In nucleic acids, there is a phosphoric acid (V) residue - H3PO4 It gives nucleic acid molecules: ❑ acidic properties ❑ a negative charge through cleavage of the hydrogen cation (proton) H+.  DNA structure Deoxyribonucleoside Nitrogenous base: C, G, A, T Sugar: deoxyribose In a nitrogenous base, the nitrogen atom is linked to a carbon atom of the sugar residue by an N-glycosidic bond The C1 carbon of the sugar is bonded to: ❑ the N1 nitrogen in pyrimidines ❑ the N9 nitrogen in purines  DNA structure Deoxynucleotide Nitrogenous base: C, G, A, T Sugar: deoxyribose Phosphate group: via a phosphodiester bond with the 5' carbon in the sugar ring phosphodiester bond N-glycosidic bond Nucleosides and nucleotides in DNA Base Nucleoside Nucleotide Cytosine (C) Deoxycytidine Deoxycytidine 5'-monophosphate (dCMP) Guanine (G) Deoxyguanosine Deoxyguanosine 5'-monophosphate (dGMP) Adenine (A) Deoxyadenosine Deoxyadenosine 5'-monophosphate (dAMP) Thymine (T) Deoxythymidine Deoxythymidine 5'-monophosphate (dTMP)  DNA structure The substrates in nucleic acid synthesis are nucleoside triphosphates  Primary structure of DNA Information is contained according to the order of the nitrogenous bases in the nucleotides (C, G, A, T) The DNA polynucleotide chain is formed by phosphodiester linkages with the 5’ and 3’ deoxyribose carbon atoms of neighbouring nucleotides, respectively The DNA chain has two different ends: ❑ the 5’ end terminated by a phosphate group ❑ the 3’ end terminated by a hydroxyl group  Primary structure of DNA The chemical differences in the phosphodiester bonds at the 5’ carbon of one nucleotide and at the 3’ carbon of the adjacent nucleotide confer polarity (directionality) of the DNA molecule The sequence of bases is written in the 5’ → 3’ direction (end of 5') ACTG (end of 3')  Primary structure of DNA end of 5’ 3’,5’-phosphodiester bonds end of 3’  Secondary structure of DNA DNA exists in a double-stranded form (dsDNA), which is composed of two complementary chains The nitrogenous bases are oriented towards the inside of the double strand and form base pairs linked by hydrogen bonds as such: A=T and G  C The structures formed by hydrogen bonds between nitrogenous bases are referred to as Watson-Crick-type base pairs or complementary base pairing The sugar and phosphate residues linked to each other by 3’,5’-phosphodiester bonds are on the outside of the chain Hydrogen bonds  Secondary structure of DNA Two complementary DNA chains wrap around a common axis to form a double helix In the DNA double helix: ❑ one strand runs in the 5’ → 3’ direction ❑ another strand runs in the 3’ → 5’ direction ❑ this arrangement is referred to as antiparallel Thus, the DNA double helix has two different orientations: ❑ one from the 5’ end to the 3’ end (5’ → 3’) ❑ another from the 3’ end to the 5’ end (3’ → 5’) Secondary structure of DNA  Secondary structure of DNA The distance between consecutive nucleotides in the DNA helix is 0.34 nm (3.4Å) There are 10 base pairs per twist of the DNA helix The twist of the helix in the DNA helix is 3.4 nm (34Å) Two depressions can be distinguished on the surface of the DNA helix, which are called the major and minor grooves These grooves are formed because the bonds connecting the base and the sugar of complementary bases (glycosidic linkages) do not lie exactly opposite to each other Secondary structure of DNA Distance between C1 atoms of two chains: 1.1 nm A-T: 1.11nm G-C: 1.08 nm Between nucleotides: 0.34 nm  Histones Two types of histones are involved in the construction of DNA : ❑ core histones ❑ linker histones The core histones are H2A, H2B, H3, and H4 Interactions between H2A and H2B, as well as, H3 and H4 lead to the formation of two dimers and then an octamer Each core histone has a long amino acid "tail" at the N terminus that extends beyond the core particle of the nucleosome The linker histones, H1 and H5, hold the DNA strands together Histones  Nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes The nucleosome core is made up of: ❑ eight histones (core histones), i.e. two molecules of H2A, H2B, H3, and H4 (histone octamer) ❑ a section of DNA ~200 nucleotide pairs long wound around the core  Chromatosome A chromatosome is a structural unit of chromatin consisting of a nucleosome and histones H1/H5 (linker histones) Histones H1/H5 are responsible for the formation and stabilization of the chromatosome Chromatosomes are connected by a ~50 nucleotide pair-long DNA linker  The 30-nm fiber The 30 nm fiber (filament) is a higher-order structure of which the presence of histones H1/H5 is important to form The resulting chromatin fiber has a diameter of about 30-nm and contains six nucleosomes per turn The packing of nucleosomes in the 30-nm fiber allows the DNA contained within it to be shortened by about 40 times Chromosome loops and domains The 30-nm fiber together with the nuclear matrix forms chromosome loops Chromosome loops are established by special non-histone proteins that bind to specific DNA sequences creating a clamp at the base of each loop Chromosome loops are then folded into looped domains Chromosome loops and domains DNA packaging in chromosomes Levels of DNA packaging in chromosomes (diameter): ❑ double-stranded DNA helix (2 nm) ❑ Nucleosome - DNA wrapped around histones (11 nm) ❑ 30 nm fiber (30 nm) ❑ loops (in dispersed form - 300 nm) ❑ domains (in condensed form - 700 nm) ❑ mitotic chromosome (1400 nm) A metaphase chromosome is 10,000 times shorter than an extended DNA helix DNA packaging in chromosomes  RNA structure Ribonucleoside Nitrogen bases: G, C, A, U Sugar: ribose In a nitrogenous base, the nitrogen atom is linked to the carbon atom of the sugar residue by an N-glycosidic bond Each C1 carbon of the pentose ring is bonded: ❑ in pyrimidines to the N1 nitrogen ❑ in purines to the N9 nitrogen  RNA structure Ribonucleotide Nitrogen bases: G, C, A, U Sugar: ribose Phosphate group: a phosphodiester bond formed with the 5' carbon of the sugar ring Phosphodiester bonds in RNA are less stable than those in DNA Nucleosides and nucleotides in RNA Base Nucleoside Nucleotide Cytosine (C) Cytidine Cytidine 5'-monophospharane (CMP) Guanine (G) Guanosine Guanosine 5′-monophosphate (GMP) Adenine (A) Adenosine Adenosine 5'-monophosphate (AMP) Uracil (U) Uridine Uridine 5'-monophosphate (UMP)  Primary structure of RNA The information in the primary structure of RNA is contained according to the order of the nitrogenous bases within the nucleotides (G, C, A, U) The RNA polynucleotide chain is formed by phosphodiester linkages between the 5’ and 3’ ribose carbon atoms of neighbouring nucleotides The RNA chain has two different ends: ❑ the 5’ end terminated by a phosphate group ❑ the 3’ end terminated by a hydroxyl group  Primary structure of RNA end of 5’ 3’,5’-phosphodiester bonds end of 3’  Secondary structure of RNA The secondary structure of RNA is formed by the folding of single-stranded RNA into motifs, such as: ❑ hairpin loops ❑ stems (helices) ❑ internal-loops ❑ bulges Secondary structure of RNA  Secondary structure of RNA The double-stranded regions of RNA form an A-form helix The formation of a B-form double-stranded helix typical of DNA molecules is predominantly prevented in RNA molecules due to having an -OH group on the 2' carbon of the ribose Regular A-type double-stranded RNA helices with Watson-Crick base pairs (A-U, G-C) have a minor and major groove Proteins bind to RNA in the minor groove region The structural diversity of artificial genetic polymers, Anasova, et al. 2015 Secondary structure of RNA Major groove: deep/short Minor groove: shallow/wide  Secondary structure of RNA In double-stranded RNA helices there are: ❑ canonical pairs of two bases (Watson-Crick type) ❑ non-canonical pairs of two bases (so-called, "non-Watson- Crick") ❑ non-canonical pairs of three bases More than 20 types of non-canonical pairs of bases connected by two, three or more hydrogen bonds have been characterized Canonical pairs of two bases Non-canonical pairs of two bases Non-canonical pairs of three bases  Secondary structure of RNA Some RNA molecules (transfer RNA (tRNA) and ribosomal RNA (rRNA)) contain modified nitrogenous bases (e.g., dihydrouridine, pseudouridine, inosine, etc.) Modifications can be: ❑ simple (e.g., methylation) ❑ complex (e.g., leading to complete reorganization of the ring) Secondary structure of RNA  Tertiary structure of RNA Tertiary structure of RNA is formed by long-range interactions formed by: ❑ arm stems with other arm stems ❑ non-canonical base pairs and triplets (loops and bulges) forming pairs other than Watson-Crick ❑ binding of basic proteins or ions that neutralize the negative charge of RNA Tertiary structure of RNA  Types of RNA Ribonucleic acids are divided into: ❑ coding; ❑ messenger RNA (mRNA) ❑ non-coding (functional); ❑ transfer RNA (tRNA) ❑ ribosomal RNA (rRNA) ❑ short non-coding RNA (sncRNA), less than 200 nucleotides ❑ long non-coding RNA (lncRNA), more than 200 nucleotides Types of RNA  mRNA Messenger RNA (mRNA): ❑ is produced in the nucleus ❑ pre-mRNA is produced, containing exons (coding sequence) and introns (non-coding sequence) ❑ pre-mRNA undergoes a multi-stage maturation process ❑ mRNA migrates to ribosomes in the cytoplasm mRNA  tRNA Transport RNA (tRNA): ❑ these are small molecules (from 63 to 94 base pairs) ❑ they occur mainly in the cytoplasm of cells ❑ they constitute 10 - 15% of the total RNA present in the cell ❑ there are 60 types of tRNA in eukaryotic cells tRNA performs two basic functions related to the protein synthesis process: ❑ recognizes the correct amino acid ❑ transfers the appropriate amino acid to the polypeptide chain created on the ribosome  Primary structure of tRNA Primary structure of tRNA: ❑ nucleotides with canonical nitrogenous bases ❑ nucleotides with modified nitrogenous bases Modified bases in tRNA: ❑ dihydrourydine - UH2 ❑ pseudourydine - Ψ ❑ inosine - I ❑ methylguanosine - mG ❑ ribotidine - T ❑ N6-isopentenyl adenosine - I6A ❑ 2(4)-thiouridyne - s2(4)U  Secondary structure of tRNA Secondary structure of tRNA: ❑ three loops - T (TψC loop), D (D loop) and anticodon loop ❑ three stems (double-stranded areas, helices) - T, D and anticodon ❑ acceptor arm Acceptor arm ❑ additional arm (variable loop) Variable loop  Secondary structure of tRNA The T-loop (TψC), also known as the pseudouridine loop: ❑ contains pseudouridine (Ψ) ❑ is involved in the recognition of ribosomes by tRNA ❑ is the site of tRNA binding to the ribosome ❑ temporarily immobilizes tRNA during the process of protein biosynthesis  Secondary structure of tRNA The D-loop (DHU) also known as the dihydrouridine loop: ❑ contains dihydrouridine (UH 2) ❑ recognizes aminoacyl-tRNA synthetases; enzymes responsible for attaching the appropriate amino acids to the appropriate tRNA molecules ❑ aminoacyl‐tRNA synthetases catalyze the reaction of creating aminoacyl‐tRNA  Secondary structure of tRNA The anticodon loop: ❑ contains a 7-nucleotide sequence, including three nucleotides that form the anticodon ❑ the anticodon binds to the bases of complementary with three nucleotides found in mRNA (i.e., the codon)  Secondary structure of tRNA Stems (double-stranded regions, helixes) within tRNA: ❑ the T stem is a short segment of five base pairs ❑ the D stem is a short segment of three or four base pairs ❑ the anticodon stem is a short-paired segment of five base pairs  Secondary structure of tRNA Acceptor arm: ❑ is formed by pairing seven base pairs ❑ the shorter 5' end always ends with a guanine nucleotide ❑ the longer 3' end always ends with the CCA sequence ❑ an amino acid is attached to the adenine nucleotide of the 3' arm, which is then transported by tRNA to mRNA in the process of protein synthesis  Secondary structure of tRNA Additional (variable) arm: ❑ small - contains 2-3 nucleotides ❑ large - contains 13-21 nucleotides ❑ can form a paired section (up to seven base pairs) o performs auxiliary functions Secondary structure of tRNA  Tertiary structure of tRNA The tertiary structure of tRNA is formed by long-range interactions In a tRNA molecule: ❑ the acceptor arm/stem (seven base pairs) interacts coaxially with the T-stem (five base pairs) forming one double helix (12 base pairs) ❑ the D-stem interacts coaxially with the anticodon stem (AS), forming a second double helix ❑ both helices form arms arranged relative to each other in such a way that they resemble the shape of the letter L  Tertiary structure of tRNA The acceptor arm is located on the opposite side to the amino acid attachment site anticodon loop anticodon  rRNA Ribosomal RNA (rRNA): ❑ is the main component of the ribosome ❑ constitutes 80% of RNA in a cell ❑ is synthesized and stored in the nucleolus ❑ consists of single-stranded chains and double-stranded helices ❑ is a ribozyme responsible for catalyzing protein synthesis in ribosomes  rRNA rRNA is found in: ❑ the large ribosomal subunit in the form of 28S, 5.8S, and 5S rRNA ❑ the small ribosomal subunit in the form of 18S rRNA The large ribosomal subunit contains about 49 ribosomal proteins The small ribosomal subunit contains about 33 ribosomal proteins The eukaryotic ribosome  rRNA rRNA active center proteins  Catalytic properties of RNA RNA molecules that are called ribozymes can catalyse a range of chemical reactions that occur in cells Often ribozymes are autocatalytic and therefore, undergo self- modification Most RNA is found in RNA-protein complexes called ribonucleoproteins (RNPs)  Catalytic properties of RNA In some RNPs, the catalytic function is performed by RNA, not protein In some RNPs, the catalytic function is performed by protein. Whereas, the RNA: ❑ guides the protein enzyme to the right place ❑ permits the enzyme to bind to the substrate It is likely that most RNA-catalyzed reactions occur with participation of proteins more or less  Roles of RNPs Step in the Gene RNP Expression Composition Function Pathway Telomerase DNA replication Telomerase RNA and Adds telomeres to the protein (Reverse ends of chromosomes Transcriptase) during DNA replication Spliceosome RNA maturation A range of snRNAs and Removal of introns from about 200 proteins pre-mRNA in the nucleus RNase P Maturation of RNA RNA and about 10 Formation of the 5' end H1 proteins of mature tRNA molecules Ribosome Translation Four rRNAs and about Synthesis of peptides, 88 ribosomal proteins polypeptides and proteins RNase P Comparison of DNA and RNA  Literature Essential Cell Biology by Alberts, et al., 6th Edition Chapter 5: DNA and chromosomes