RNA structure and function

• RNA has primary, secondary and tertiary structures
• Primary structure: Polynucleotide
• Secondary structure: Single and double stranded with loops
• RNA structure depends on its function
• RNA is structurally more varied than DNA

Biological Functions of RNA

• Realizes information, and can store it in some viruses
• mRNA (messenger RNA) is synthesized during transcription and delivers copied information to the cytoplasm for protein synthesis
• tRNA (transfer RNA) binds activated amino acids and delivers them to ribosomes for protein synthesis; there are at least 20 different types
• Isoacceptor tRNA exists in eukaryotic cells, binds to the same amino acids and regulates the speed and intensity of protein synthesis
• rRNA (ribosomal RNA) is part of the ribosomal complex, catalyses formation of peptide bonds, and controls mRNA binding
• Regulatory RNAs are noncoding RNA molecules; they impact differentiation and development through epigenetic regulation
  • Small nuclear RNA (snRNA)
  • Micro RNA (miRNA)
  • Small interfering RNA (siRNA)
  • Long non-coding RNA (lncRNA)

DNA Replication

• DNA replication duplicates DNA molecules during the S phase of the cell cycle in eukaryotes, and also in prokaryotic cells
• Replication starts with binary fission
• It utilizes complementary base-pairing, catalyzed by specific enzymes and proteins
• Semiconservative replication: one strand from old gives the newly synthesized strand of DNA
• Replication initiates at the origin of replication
• Requires an initiation protein, and contains a specific motive of DNA nucleotide sequence

DNA Replication Initiation

• DNA double-helix uncoils via the enzyme helicase, which cleaves hydrogen bonds between nucleotides
• This cleaving creates a Y shaped replication fork
• Topoisomerases prevent DNA from supercoiling
• Single strand binding proteins prevent reformation of the double helix

Leading and Lagging Strands

• Leading strand synthesizes new continuous DNA; Lagging strand discontinuous synthesis of new DNA (Okazaki fragments).
• DNA synthesis starts with RNA primers, which are 10 to 12 nucleotides long, and complementary to the single stranded DNA
• Primase, a part of the Pol α-primase complex (in eukaryotes), ensures this process
• Polymerization of the strand starts from the primer and is ensured by DNA polymerase, which is part of the Pol α-primase complex

DNA Synthesis Direction

• The new strand is synthesized from free dNTPs (deoxyribonucleotide triphosphate) in the 5' to 3' direction
• Free dNTP can only attach to the free 3' OH group of pentose
• Pyrophosphate is released, and its hydrolysis generates energy for polymerization

Elongation

• DNA is synthesized from leading and lagging strands by different polymerases
• DNA polymerase epsilon (ε) synthesizes the new stand from the leading stand; also does error repair
• DNA polymerase delta (δ) elongates the lagging strand; also does error repair
• RNase H recognizes and degrades RNA primers after the new DNA strand is synthesized
• DNA polymerase beta (β) fills in empty spaces
• DNA ligase ligates the 5' phosphate of a nucleotide with the 3' OH group

Protein functions

• Initiation proteins recognize the origin of replication
• Helicase unwinds DNA
• Single-strand binding proteins stabilize single-stranded DNA
• DNA topoisomerase reduces super helical tension
• RNase H removes the RNA primer
• DNA ligase connects discontinuous DNA fragments
• DNA polymerase α/primase starts synthesis of the leading strand and each Okazaki fragment
• DNA polymerase δ replicates the lagging strand.
• DNA polymerase ε replicates the leading strand

Telomeres and End Replication

• Telomeres are the ends of linear DNA containing repeated nucleotide sequences specific to species(Human telomeres contain the sequence TTAGGG and there are 100-1000 repeats)
• The 3' end of telomeres are single stranded and form a cap
• Results in a problem arising along the lagging strand after replication
• Enzyme telomerase contains several subunits, which includes a integral RNA component
• Acts as a template for a telomere cap synthesis on a single strand
• Normal DNA replication, replication error, and oxidative DNA damage affects telomere length

Prokaryotes vs. Eukaryotes DNA Replication

• Prokaryotes

  • Location: Cytoplasm
  • Origin of replication: One, 100-200 nt
  • Replication forks: Two bi-directional -Initiation: Two proteins
  • Okazaki fragments: Long, 1000-2000 nt -Speed: Fast – 2000 nt/sec

• Eukaryotes

  • Location: Nucleus
  • Origin of replication: Multiple (>1000), each ~150 nt
  • Replication forks: Parallel, many origins of replication -Initiation: Multi-subunit proteins
  • Okazaki fragments: Short, 100-200 nt
  • Speed: Slow – 100 nt/sec

Nucleus

• The nucleus houses linear DNA molecules and is known as “controlling centre” of a cell
• The nuclear envelope is a double-layered membrane complex
• Separates the nucleus from cytoplasm in eukaryotic cells and mRNA protein synthesis

Nuclear Structure

• Outer nuclear membrane: 7-8 nm thick, linked with ER and contains ribosomes
• Inner nuclear membrane: 7-8 nm thick, contains integral membrane that connect to chromatin and nuclear lamina proteins
• Perinuclear space: 10-30 nm wide, fluid-filled compartment that is a continuation of the ER lumen

Nuclear Pore Complex (NPC)

• Part of nuclear envelope and forms channel
• Made from proteins called nucleoporins (Nups)
• Facilitates exchange between the nucleus and cytoplasm which is dependent on cell activity with 30-50 pores per 1 mm2

Nuclear Components

• Central transporter: Connects nucleoplasm and cytoplasm for macromolecular exchange assisting transport assisted via nuclear transport receptors
• Spoke: 8 cylindrical nucleoporin groups around transporter
• Small molecules/ions: Undergo passive transport
• Macromolecules: Active transport assisted by nuclear receptors
• Importin β: Binds importing macromolecules
• Exportin: Binds exporting macromolecules

Linkers of Nucleoskeleton and Cytoskeleton (LINC) Complex

• Protein complex of Nesprin and SUN proteins in mammals
• Positions nucleus, coordinates nuclear/cytoplasmic activities, and centrosome near nucleus
• Involved in mechanical force transmission from cytoskeleton

Nuclear Lamina

• Structure attached to the inner nuclear membrane via proteins, protein complexes, and chromatin
• Composed of proteins called nuclear lamins
  • A-type lamins: A and C isoforms, encoded by LMNA gene
  • B-type lamins: Encoded by LMNB1 and LMNB genes
• Binds to the integral inner nuclear membrane proteins
• Nuclear lamins forms intermediate filaments specific to the nucleus
• Involved in DNA replication, transcription, nuclear/chromatin organization, cell-cycle regulation, cell development, nuclear migration, and apoptosis
• Changes to DNA coding region for Lamins and lamina proteins leads to inherited disorders called laminopathies

Emery-Dreifuss Muscular Dystrophy

• Symptoms: Loss of joint motion, slow progressive muscle weakness, and cardiac disease
• Molecular Mechanism: Dysfunction of the LINC/emerin/Lamin protein complex of the nuclear envelope
• Emerin is integral protein of inner membrane interacting with lamina and organizing cell division; helps to stabilize the nuclear membrane from mechanical muscle stress

Nucleoplasm (Karyoplasm)

• Protoplasm of the nucelus and has suspened chromatin material, nuecleolus, and other particulate elements
• Nucleoproteins are the major component
  • Proteins + Nucleic acids = Nucleoproteins

Proteins

• Non-histones: Small acidic proteins, phosphoproteins, and enzymes, but low counts overall
• Histones: Mainly basic amino acids (arginine, lysine, histidine) and positive charge
  • Types: H1, H2A, H2B, H3, H4 (pack DNA).

Chromatin

• The majority of DNA in eukaryotes and is composed of nucleosomes
• Lowest level of chromatin organization. Nucleosome comprised of eight histone molecules(H2A, H2B, H3, H4
• Histone octamere 1.65x is wrapped by 147 nucleotide long negatively charged DNA; 60 nucleotide linker b/w octameres
• Linker DNA binds tightly with histone H1 closes the nucleosome and forms chroamatosome.

Interphase Chromosome Structure

• DNA plus nucleoproteins that has long coiled linear structure
• Chromosomes occupy specific space called chromosomal territories
• Each chromosome divided into topological associated domains (TAD).
• Repressed transcription activity TADS associated nuclear lamina, while active reside nuclear interior

Euchromatin

• Nucleosomes are loosely assembled regions with high gene expressions
• Makes up 5% of chromatin and is early replicative

Heterochromatin

• Nucleosomes form large assemblies, very limited or no gene expression
• Associated with telomere and centromere regions and chromosome
• Makes up 95%
• Late replicative and found only in eukaryotic cells
• Types include:
  • Constitutive: Repetitive sequences with inactive DNA and serves as a chromosome element abundant in chromosomes 1,9,16,19 and the Y chromosome
  • Facultative: Becomes heterochromatic in certain cells to make the inactive body
• Quote: Heterochromatin is methylated and histones are hypo-acetylated (inactive). It does not participate in genetics

Function of Heterochromatin

• Centromere function
• Organization of nuclear domains
• Gene repression(epigenetic regulation)

Metaphase Chromosome Structure

• Tight fiber produces the chromatid of a chromosome during S-Phase forming two identical chromatids
• Formed via condensins for condensation and stabilization, and cohesion to replicates for the sister chromatid contact
• Contain the centromere with proteins and chromatin forming the kinetochore attachment site for chromosome segregation
• Satellites are tamdemly repeated DNA for silent regions; second contriction is the narrowing of the chromosome with satellites
• Nucleolus organization regions is a region with coding sequence that are clustered on chromosome arms
• The ends of the DNA known as telomeres containing a repeated nucleotide sequence

Telomeres

• Species specific and human telomeres contain the repeat TTAGGG for 230 kb and single stranded for 100-200 nucleotides
• POT1 binds single stranded repeats

Telomere Function

• Serves as a counting mechanism and capping function that leads to cellular instability
• Increased telomerase activity in stem/embyronic cells and most cancer cells