BIO 5099: Molecular Biology for Computer Scientists (et al) Lecture 15: Being a Eukaryote: From DNA to Protein, A Tour of the Eukaryotic Cell. Christiaan van Woudenberg Being A Eukaryote Basic eukaryotes have: Plasma membrane cell boundary, retains cytoplasm, selective barrier. Cytoplasm portion interior to the plasma membrane, fluid contents of the cell. Cytoskeleton microfilaments & microtubules, holds everything in place, allows motility within cell. Organelles nucleus, Golgi, endoplasmic reticulum, vesicles, mitochondria, etc. What do they look like? Eukaryotic Cells nucleolus rough endoplasmic reticulum mitochondrion Golgi complex 1
Eukaryotic Cell Organelles Prokaryote vs Eukaryote Structure Cytoplasmic membrane Nucleus containing a clear membrane surrounding DNA Ribosomes Cell Wall Internal organelles Endoplasmic reticulum Golgi complex Mitochondria Chloroplasts Vacuoles Prok. 70S Euk. 80S,,, DNA Structure Human DNA is 3 billion bp, 30,000 genes, 200,000x longer than average cell width. Wound around protein complexes called histones. Unused portions condensed into heterochromatin. Unpacked euchromatin faciliates transcription, replication. Compaction depends on cell cycle stage. 2
Histones Protein octamer that winds up 146bp of DNA into nucleosome to give us 30 nm filament. 7:1 packing ratio on histone, 40:1 on 30 nm filament. High arginine/lysine ratios. Highly conserved across eukaryote species. DNA Domains Contain 100kbp loops, anchored to nuclear scaffold by ATrich sequences. Chromosomes Only take their fully compact form during cell division, which we ll discuss later. Karyotype allows us to visualize abnormalities. Daughter chromatids are 3
Chromosomes Daughter chromatids are bound at the centromere. Kinetochore is where microtubules attach during mitosis (more later). Telomeres at ends of chromosome are TTAGGG sequence repeats (5003000 times), maintained by telomerases. Telomere shortening is important in aging. telomere Visualization Techniques Fluorescent InSitu Visualization (FISH) uses fluorescence markers to highlight particular chromosomes, structures, genes or sequences. Q (AT rich) and R (GC rich) bands are stained to prepare karyotypes. Sample Human Karyotype 4
Eukaryotic Nucleus Contains all genetic material as chromatin, segregated from the rest of the cell by the nuclear envelope. Inner membrane is nuclear lamina. Outer membrane extends to endoplasmic reticulum (more soon). Nucleolus is site of ribosomal RNA synthesis. Structure maintained by nuclear matrix. Studded with nuclear pores. Eukaryotic Nucleus Import of: RNA polymerases DNA polymerases Ribosomal proteins Histones Transcription factors Export of: Ribosome subunits trnas mrnas snrnas Nuclear Pores Serves as a waterfilled channel. Mechanism for moving molecules across nuclear membrane. Ring of 8 multidomain protein subunits. Allows passive diffusion of small molecules and ions. Proteins, RNAs, other larger molecules are actively transported. Opens in response to Ca 2 ions. 5
Making Proteins (A) PROCARYOTES DNA mrna protein TRANSCRIPTION TRANSLATION (B) EUCARYOTES cytoplasm nucleus introns exons DNA gene TRANSCRIPTION primary RNA transcript RNA cap ADD 5' CAP AND POLY(A) TAIL AAAA RNA SPLICING mrna AAAA EXPORT mrna protein AAAA TRANSLATION The Protein Pathway Proteins may be destined for: Cytoplasm, Intracellular organelles, Plasma membrane, Extracellular export. A variety of mechanisms are used depending on target destination. The entire set of structures is called the endomembrane system. Endomembrane System Composed of: Endoplasmic reticulum (rough and smooth) Golgi Complex Other vesicles such as: Vacuoles Lysozomes Peroxisomes 6
Smooth Endoplasmic Reticulum Synthesis of steroid hormones. Degradation and detoxification in concert with lysosomes and peroxisomes, carried out by cytochromes. Glycogen metabolism in liver cells. Regulates Ca 2 ion release into cytosol important for muscle contraction, other regulatory elements. Rough Endoplasmic Reticulum Rough due to membranebound ribosomes. 13 million ribosomes per liver cell. Synthesis of proteins to: Be secreted outside the cell, Be membrane bound, Remain in the ER or Golgi complex. is done in the rough endoplasmic reticulum. Glycosylation of proteins. How does this work? Targeting Protein Synthesis Proteins destined for cytosol, nucleus are synthesized by free ribosomes in cytosol. Ribosome translates hydrophilic signal peptide, stops translation, is directed to rough endoplasmic reticulum to continue. A signal recognition particle (SRP) binds peptide sequnce with receptor on rough ER to continue translation. Proteins synthesized by ribosomes on surface are injected into interior (lumen) through the SRP channel. 7
Signal Recognition Complex mrna trna 3' 5' Ribosome sampling Signal sequence recognition signal sequence SRP Elongation arrest 3' 5' GTP Dissociation Pi Targeting 3' 5' 3' 5' Cytosol ER Lumen SRP Receptor Translocon, ribosome receptor Translocation Golgi Complex Vesicles bud from rough ER and merge into first layer of Golgi complex. Has several layers called cisternae, arranged like a stack of pancakes. Cis face towards ER, trans face towards cell membrane. Completes synthesis of some proteins, membrane lipids. Golgi Complex 8
More Golgi Complex Proteins pass through stacks of the Golgi embedded in vesicles that bud, then merge into next layer. Different parts of Golgi modify protein sequences: Trim off signal peptides from sequence, Hydroxylation of amino acid residues, Glycosylation to create polysaccharides, and more! Protein Secretion newly synthesized soluble proteins for constitutive secretion newly synthesized plasma membrane lipids unregulated membrane fusion newly synthesized plasma membrane protein CONSTITUTIVE SECRETION plasma membrane trans Golgi network CYTOSOL signal transduction signal such as hormone or neurotransmitter Golgi apparatus secretory vesicle storing secretory proteins regulated membrane fusion REGULATED SECRETION Lysosomes Contain acid hydrolytic digestive enzymes made in the rough ER. Vary in shape and size from cell to cell, from 25nm to 1um (40x size variation). Vesicles is formed by budding from Golgi complex. Function to degrade proteins, enzymes, etc. Lysosome engulfing a mitochondrion. 9
Peroxisomes Vesicles that contain crystalline core of oxidative enzymes. 0.1 to 1.0 um in diameter. Contain 50 or more enzymes with varied functions: Oxidation of fatty acids, Cholesterol synthesis in liver, Not synthesized from Golgi made from other peroxisomes. Mitochondria From the inside out: The matrix contains mito DNA, ribosomes, other proteins. Also where Krebs cycle happens. Inner membrane is site of electron transport chain, also ATP synthase. Inner membrane has folds called cristae to allow for additional capacity for electron transport chain and ATP synthesis. H ions are pumped into intermembrane space by ETC. ATP Synthase allows H back into matrix to create ATP. Outer membrane separates from cytosol. Cytoskeleton Provides: Structural support Intracellular transport Contractility and motility Spatial organization Highly dynamic structures, always moving, always changing. 10
Cytoskeleton Components Intermediate filaments form nuclear lamina, gives cell mechanical strength. Microtubules long, rigid, made of tubulin. Microfilaments long, flexible, made of actin. 25 mm 25 mm 25 mm INTERMEDIATE FILAMENTS MICROTUBULES ACTIN FILAMENTS 25 nm 25 nm 25 nm Intermediate Filaments 10 nm wide, often radiate in spiral pattern. Heterogeneous encoded by at least 50 genes. Usually formed as a tetramer of two dimers. Highly resistant to stretching. Provide mechanical stability, tissuespecific functions. Microtubules Made of noncovalently bound tubulin dimers, 24 nm wide, easy to make/break. Form cilia and flagella. Polarity in tubulin dimers gives filaments direction. Kinesin moves towards plus end. Cytoplasmic dynein moves to minus end. Form mitotic spindle during mitosis, attach to centrosome. 11
Microfilaments Made of double strand of actin chains, 8nm diameter. Play a role in almost all cell s motility processes. With myosin, form muscles. 12