Molecular Cell Biology Core Concept Master Cheat Sheet O1: Introdu ction Molecular Cell Biology •
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03: Cells: The Chemical Fo unda tions
Molecular Biology is the study of the replication, transcription, & translation of genetic material within a cell. Manipulation of these processes is also known as molecular biology or recombinant DNA techniques. Macromolecules- there are four main classes of macromolecules: lipids, proteins, carbohydrates, and nucleic acids. Deoxyribonucleic acid (DNA)- double helix chains of paired bases containing thymine, cytosine, guanine, and adenine. Ribonucleic acid (RNA)- the intermediate between DNA and proteins.Proteins- chains chains of amino acids coded for by genes in the DNA.
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Transcritpion: DNA is transcribed into RNA and special pairing of bases dictates which sequence is made.
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Organic chemicals: contain covalently bonded carbon backbones. Isomers: chemicals with the same molecular formula but different structural formulas. Buffers: solutions, which resist changes in pH upon addition of small amounts of acid or base. Electrolytes: chemicals that release ions into solutions. pH : pH represents the concentration of hydrogen ions [H+] in solution (scale 1-14, pH 7=neutral). 7=neutral). pH = -log [H+] Acids: release H+ into solution, pH < 7 Bases: release OH- that can combine with H+ to make water, pH > 7 Enzymes: proteins that serve as catalysts for biochemical reactions. Entropy: a measure of a system's degree of disorder. It increases with increasing disorder. Laws of thermodynamics: First Law: The total energy of the universe is always conserved. Energy can neither be created nor destroyed. Second Law: The universe tends towards maximum disorder; the direction of all spontaneous processes serves to increase the entropy of a system plus its surroundings. Energy of activation (Ea) = the free energy necessary to start a reaction. Enzymes act as catalysts to lower E a, but they do not change
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02: The Cell • •
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Cell: The smallest unit of an organism that can live independently. Cell Theory: an explanation of the role of cells in relationship to living things. · Prokaryotic Cell: The smallest unit of life for prokaryotic organisms such as bacteria and other microbes. Eukaryotic Cell: The smallest unit of life for eukaryotic organisms such as animals. Nucleolus: a “tiny nucleus” inside the nucleus, which contains RNA. Nuclear Envelope: a protective layer, which surrounds the nucleus. Nuclear Pore: holes in the nuclear envelope that allow the nucleus to exchange information with the rest of the cell. DNA: a nucleic acid, an organic macromolecule. RNA: a nucleic acid, an organic macromolecule. Cytoplasm: the liquid space inside of a cell. Organelles: specialized cell structures, which perform specific functions. Cell Membrane: a protective bilayer of lipids, which allows the cell to maintain a stable internal environment. environment.
No Enzyme Reactants
+ Enzyme
Ea
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Products
04: The Macromo lecules Macromolecule: A molecule having a molecular weight in the range of a few thousands to many millions. • Functional group: The specific atom or group of atoms that confers a particular chemical property on a biomolecule. • Organic Compounds: Molecules containing covalently bonded carbon backbones are called organic compounds. • Monosaccharide: A carbohydrate consisting of a single sugar unit. • Disaccharide: A carbohydrate consisiting of two covalently joined monosaccharide units. • Polysaccharide: A linear or branched polymer of monosaccharide units linked by glycosidic bonds. •
Potential Lysosome
Nucleus
ABCD BACD CABD DABC
Ribosomes Smooth Endoplasmic Reticulum
Mitochondria
Golgi Apparatus
ABDC BADC CADB DACB Rough Endoplasmic Reticulum Plasma Membrane
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Monomers A,B,C,D
05: Protein Structure and Function • •
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07: DNA Structure and Function
Antibody: Antibody : A specific protein that interacts with a foreign substance (antigen) (antigen) in a specific way. Beta-sheet (ß-sheet): A sheet like structure formed by the interaction between two or more extended polypeptide chains. Cytoskeleton: The filamentous skeleton, formed in the eukaryotic cytoplasm that is largely responsible for controlling cell shape. Dalton: A unit of mass equivalent to the mass of a hydrogen atom (1.66 x 10-24 g) Disulfide Bridge: A covalent linkage formed between two cysteine-SH groups either in the same polypeptide chain or in different polypeptide chains. Enzyme: A molecule, most often a protein that contains a catalytic site for a biochemical reaction. Globular protein: A folded protein that adopts an approximately globular shape. May also be called soluble proteins.
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Nucleoside: Compounds with a ribose or deoxyribose and a base. Base includes includes four types G, C, A and T (or U). Nucleotide: Compounds with a ribose or deoxyribose, a base and a phosphate group, it is the basic building unit for DNA (monomers of DNA). Pyrimidines: A type of base, including both cytosine and thymidine. Purines: A type of base, including both guanine and adenine. Tm : Melting temperature of DNA. DNA is a double helix molecule, when heated, the two strand separate from each other (melting) and the absorbance at 260 nm increases. When half of the DNA is in single strand status, the temperature is called the DNA’s Tm. Denature: The hydrogen bonds break between the two strands of the DNA molecule, can be caused by heat or pH. Renature: The denatured DNA restore the double helix structure and re-form hydrogen bonds.
Residue n+1
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Peptide bond 06: Gene Gene and Chromosomes • •
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Karyotype: A complete set of all metaphase chromosomes chromosomes in a cell. Species-specific and is used for genetic genetic diagnosis. G band and Q band: Bands shown on chromosomes after various treatments and staining technique. Same chromosome from one specieis always show same banding pattern. FISH: fluorescence in site hybridization. A fluorescence labeled DNA fragment (probe) is hybridized to a chromosome therefore a gene can be located to a chromosome. Nucleosome: A bead-string structure formed by DNA and histones, the basic DNA structure in a chromatin. Chromatin: The general structure of any chromosome, the basic units is nucleosomes. Chromatid: One-half of a replicated chromosome. Histone: structure proteins for nucleosome, including H1, H2A, H2B, H3 and H4 types. Chromosome: The unit of inheritance with the basic structure of arms, a centromere and two telomeres . Euchromatin: Chromatin region stained lightly, usually are lightly packed and transcriptionally active. Heterochromatin: Chromatin region stained dark, usually are heavily packed and transcriptionally inactive. Telomere: The DNA-protein strucuture at the ends of each chromosome plays critical role in DNA replication and aging.
08: Transcription, Transcription, Translation and The Genetic Code •
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mRNA: A messenger between DNA and protein, made from DNA template (transcription) and then directs the protein synthesis. tRNA: Transfer RNA, carry amino acid to protein synthesis site. rRNA: Structure RNA of ribosomes where proteins are synthesized. microRNA: miRNA, have mismatch to mRNA, but they bind to mRNA, leading to mRNA degredation or block mRNA translation. Template strand: The transcribed DNA strand, with sequence complementary to mRNA sequence. Non-template strand: Not transcribed, sequence is identical to mRNA, also called coding strand. Promoter: A DNA sequence having a regulatory function over the transcription of an adjacent gene, and to which RNA polymerase binds prior to transcription.
miRNA
Dicer
Degredation
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Block Translation
09: Control Control of Gene Expression in Prokaryotes •
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11: Recombinant DN A Technology •
Promoter: A DNA sequence having a regulatory function over the transcription of an adjacent gene, and to which RNA polymerase binds prior to transcription. Pribnow Box: A conserved promoter sequence located at10 position of mRNA, usually is TATAA, also called TATA box, the function is for transcription factor binding. Operon: consist of two or more adjacent coding regions that are controlled by the same transcription factors. factors . Polycistronic: The structure genes of an operon are transcribed as a single mRNA molecule containing multiple genes. Structure genes: The genes in an operon that encodes the enzymes necessary for a metabolic pathway. Regulatory genes: The genes in an operon that play regulatory roles, acting as either repressors or activators. Cis-acting element: DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression. Operator sequence and promoter sequence sequence are cis-acting elements. elements. Trans-acting Trans-acting elements: Factors that bind to the cis-acting sequences to control gene expression, for example, RNA polymerase, TFs, repressors.
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Recombinant DNA: a DNA molecule that is made from two different sources and often manipulated in a test tube and a host cell. cDNA: complementary DNA, the DNA synthesized from a mature mRNA template. cDNA is often used to clone genes. Genomic DNA: DNA isolated from organisms, containing all DNA in the genome of a cell or an organism. organism . PCR: polymerase chain reaction, a molecular biology technique using DNA polymerase, template DNA and primers to synthesize specific DNA fragments. Restriction Endonuclease: an enzyme that cuts doublestranded DNA within the molecule. DNA vector: an agent that can carry a DNA fragment into a host cell and ensure its replication and sometimes, expression. Cloning vector: A vector that carries DNA into a host cell for replication. Expression vector: A vector that carries DNA into a host cell and express the DNA into its final protein product.
Digest with same restriction restriction enzyme BamH I
Sticky ends TrpR
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T rp L
TrpE TrpD TrpC TrpB
TrpA Anneal
mRNA
mRNA
no Trp, no bind
seal with DNA ligase
Trp syn synthes thesis is
Repressor R
Recombinant DNA
12: Genomi cs 10: Regulation o f Gene Expression in Eukaryotes •
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Promoter: A DNA sequence having a regulatory function over the transcription of an adjacent gene, and to which RNA polymerase binds prior to transcription. Enhancer: a short region of DNA that can be bound with proteins to promote expression of a distal or proximal gene. TATA Box: Binds to transcription factor for regulating gene expression, usually within 30bp of the transcription start site. Basal Transcription Factor: Transcription factors that are required for all transcriptions, they unwind DNA and assist RNA polymerase to bind to transcription initiation sites. Modulatory Transcription Factor: Transcription factors that regulate time/space expression, may bind to enhancers or to promoter regions to enhance or suppress gene expression under certain conditions. DNA binding domain: A domain found in all transcription factors, used to bind DNA, inclding three types: Zinc-finger, HLH and leucine zipper. Zinc-Finger: A structure found in a number of transcription factors, has repetitive two cystein and two histidine to bind a Zn, and form a “finger” structure. Leucine-zipper: A structure found in a number of transcription factors, characterized by leucine residue at every other turn of helix, 7 amino acid apart.
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Genomics: the study of an organism's entire genome, usually starts with whole genome sequencing. BAC: bacterial artificial chromosomes, which is based on F plasmid and can tolerate large inserts, widely used in genomic DNA library construction. BAC library: large genomic DNA fragments ligated into BAC vectors. Shot gun sequencing: Genomic DNA from a BAC clone is fragmented fragmented into smaller size, typically 1kb, and inserted into a sequencing plasmid. These sequences sequences are then assembled assembled based on the overlapping fragments. Sequence assembly: a computer-based approach to put together all the genomic DNA sequence together, starting to put together a BAC sequence first. Human Genome Project (HPG): Divided into two groups based on different funding resource: public sector and Celera private sector. sector. The purpose is to obtain all genetic genetic information from humans. Functional Genomics: Use the sequence data to explore how DNA and proteins work with each other and the environment to create complex, dynamic living systems.
Procedure Overview Step 1: BAC library construction
Chromosome
Step 2: Shot gun sequencing
Step 3: Sequence assembly
Enhancer
Promoter
TATA Box
Trans Transcrip criptio tion n start starts s
BAC library
Enha Enhanc ncer er
Shot Gun Sequencing Exon
Intron
Sequence Assembly
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13: Membrane Structure •
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1 5: Cellular Energetics
Membrane Structure: Structure: Maintains and protects cellular contents, provides for electrochemical gradients, dynamic and flexible enough to allow cell shape changes and cell movements, supports membrane bound proteins, which are involved in cell signaling and growth and differentiation. Phospholipids: make up the major portion of membranes in cells. They are Amphipathic molecules, which means they have both a hydrophobic (water fearing) and a hydrophilic (water loving) component. Phospholipid Bilayer: By forming a bilayer, the hydrophobic tails can interact with each other and the hydrophilic heads can interact with water molecules in the surrounding fluid. Cell Membrane: is made up of phospholipids, glycolipids, cholesterol and proteins. A cell membrane is approximately 5 nm thick and is a fluid dynamic structure. Cholesterol: is very prominent in the membranes of cells; it inserts itself within the bilayer. Cholesterol enhances the permeability-barrier permeability-barrier properties of the cell membrane.
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Glycolysis: Glycolysis is a pathway in which a molecule of glucose is oxidized into two molecules of pyruvic acid. Glycolysis takes place in the cytosol of the cell. Electron Transport: a series of r edox reactions take place, in which one molecule is reduced (gains an electron) and the next is oxidized (gives up an electron). At each step, a Proton (H+ atom) is pumped across the mitochondrial membrane, creating the concentration gradient used by the enzyme ATP Synthase. NADH: is an important electron transporter in the respiratory chain enzyme complexes. NADH is the reduced form of NAD+, and NAD+ is the oxidized form of NADH. Oxidative Phosphorylation: involves the phosphorylation of ADP to form ATP. These reactions depend on the H+ proton gradient that is created during the events of the respiratory chain. ATP Synthase is an enzyme also known as the F0F1 ATPase. Photosynthesis: utilizes the energy from sunlight to eventually form hexose, sucrose and starch along with oxygen. These reactions begin with the absorption of light photons, and then involve electron transport and, ultimately, carbon dioxide metabolism.
14: Membrane Transport •
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Membrane Transport: Molecules that are small and hydrophobic can pass through the cell membrane by passive diffusion, down their concentration gradient i.e, oxygen and carbon dioxide. Facilitated Diffusion: is carried out by a carrier protein or channel protein and transports the molecule passively, down its concentration gradient. Active Transport: is the process by which a carrier protein, also known as a pump, facilitates the movement of a molecule against its concentration gradient. This process requires metabolic energy from ATP hydrolysis. Na+-K+ Pump: utilizes the energy released from ATP hydrolysis to transport Na+ and K+ ions against their concentration gradients. gradients. This pump is responsible for the membrane electrical gradient inside relative to outside the cell. Ion Channels: Exist in either an open or closed state. The different types of channel proteins are: (A) Voltage-gated channels, (B) Ligand-gated and (C) Nucleotide-gated channels.
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16: Intracellular Compartments •
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Intracellular Compartments: Compartments: Cells contain many special compartments, compartments, known as organelles. There are many crucial enzymatic and non-enzymatic processes necessary for cell survival that take place in or on lipid membranes. Nuclear Protein Import: Proteins that are targeted for the nucleus contain a nuclear localization signal. Nuclear import receptors bind the nuclear localization signal portion of the protein and facilitate its transport into the nucleus. Intracellular Protein Transport: There are 3 main ways in which proteins are transported within cells: (A) Gated transport, (B) Transmembrane transport and (C) Vesicular transport. Posttranslation Translocation: Proteins are imported through multi-subunit protein complexes, known as translocators; both the insertion of the protein into the translocator and the exit on the opposite side of the membrane requires energy.
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17: Intracellular Vesicular Traffic •
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19: Cell Signaling Signaling P athw ays
Molecular Mechanisms o f Coated Vesicles: Most vesicles bud off from a region of the membrane that has a unique protein coat (cage). There are 3 different types of protein coats used by cells: (A) Clathrin-coated, (B) COPI-coated and (C) COPII-coated vesicles. COPI and COPI I-Coated Vesicles: Vesicles: COPI-coated vesicles transport cargo from the cis end of the golgi complex to the endoplasmic reticulum (ER), called retrograde transport. COPII-coated COPII-coated vesicles transport cargo from the endoplasmic reticulum to the golgi complex, called anterograde transport. ER-Golgi ER-Golgi Transport: Proteins that are targeted for the golgi complex are packaged into COPII-coated vesicles. Vesicles bud from special ER regions, called RE exit sites; these sites usually do not contain ribosomes. Golgi-Lysosome Transport: Mannose 6-phosphate (M6P) is a lysosomal sorting signal. Proteins that are destined to reside in the lysosome, such as lysosmal hydrolase, are tagged with a M6P.
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Cell Communication: Cells interpret incoming signals in order to: secrete molecules, move location and proliferate into new cells. Forms of Signaling: Secreted molecules can mediate 3 forms of signaling: Paracrine - secreted molecules can diffuse a short distance in the extracellular matrix and then be taken up by adjacent cells, Autocrine - secreted molecules can act on the cell they were released from and Endocrine: molecules secreted into the bloodstream, which are then carried to the target cells and diffuse out into the tissue. G-protein Linked Cell-Surface Receptors: G-protein receptors exist in the plasma membrane of many cell types; they have an extracellular domain - where the receptor binds molecules outside of the cell and initiates the signal transduction and an intracellular domain - interact with G proteins and stimulate their activation through binding of ATP. G-protein: Shutting off the Signal: After the G-protein activates cyclic AMP, it hydrolyses GTP into GDP and is then returned to an inactive state.
18: Cellular Cellular Commun ication • • •
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Chemical signal: Cell signals are often chemicals. Paracrine signal: local signals that can only influences the neighboring cells, such as neuron transmitters. Endocrine Signal: Specialized cells release molecules (often hormones) into blood vessels of circulatory system, hormones move to distant target cells. cells . Signal receptors: often membrane proteins that can bind signal molecules from cell surface except for intracellular receptors, which is localized in cytosol. RTK: receptor tyrosine kinase, numerous typs but oftern include receptors receptors for growth factors. factors. It has intrinsic tyrosine tyrosine kinase activity and is capable of self activation by phosphorylation and dimerization. GPCR: G-protein coupled receptors, usually has 7 transmembrane transmembrane domain and has an intracellular domain to bind signal molecule and a cytosol domain to bind G protein. G Protein: A huge class of proteins that bind to guanine nucleotide GDP and GTP, usually hetertrimers and associate with GPCR or cytosol cytosol side of cell membrane. membrane. Upon signal binding by a GPCR, i t undergoes a conformational change and hydrolizes GTP to activate the downstream targets.
20: Cytoskeleton Cytoskeleton and Cellular Motility •
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Cytoskeleton: provides dynamic structure and support for cells, which is reorganized frequently, and provides and maintains cell shape. Cytoskeleton Cytoskeleton Functions: facilitates intracellular movements including: Vesicle Transport, and Chromosome Segregation. Actin Filaments: also known as microfilaments are organized into bundles just underneath the plasma membrane. Actin filaments, along with motor proteins such as myosin, are reorganized as cells change their shape and move. Intermediate Filaments: are made up of various intermediate filament proteins including: keratins, vi mentin, neurofilament neurofilament proteins and nuclear lamins, and share the same long rope-like structure and are prominent around the nucleus and extend out towards the periphery of the cell. Microtubules: are long, hollow tube-like cylinders, which are made up of the protein tubulin, and are involved in vesicle and organelle movement within cells. ·
Signal Reception
Transduction
Receptor Cell membrane
Transducer Effector Response
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21: The Cell Cycle and P rogrammed Cell Death •
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23: Cancer •
Cell Cycle: The Cell Cycle is made up of 4 distinct phases: (1) Gap 1 (G1), (2) S Phase, (3) Gap 2 (G2) and (4) M Phase (Mitosis). G1 : In cells that are actively cycling through the cell cycle, G1 is when the cell monitors its environment and size. During this time the cell grows. S Phase: In order for a cell to divide successfully, it must copy its entire DNA to be able to supply 2 daughter cells. During S phase, (synthesis phase) a cell must copy its chromosome and the entire DNA it contains. Mitosis: Mitosis involves the dividing of the nucleus and the surrounding cell. Mitosis occurs after the successful replication of the cell’s DNA. During mitosis, the parent DNA and copied DNA must be separated. Following this event, the cytosol and cell membrane divide during cytokinesis. Programmed Cell Death: Death: Programmed cell death is a mechanism used by the body to remove unwanted cells as part of normal development, injury and disease. It is the deliberate suicide of a cell that is highly regulated and quite different from necrosis, which includes inflammation and accidental cell death. A family of proteolytic enzymes called caspases mediates programmed cell death. These caspases are stored in cells as inactive pre-caspases
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Cancer: The general name for over 100 medical conditions involving uncontrolled and dangerous cell growth. Oncogenesis: process of initiation of tumors (cancer) in an organism. organism. Tumor: tissue composed of cells that deviate from normal program of cell division and differentiation. Benign tumor: tumor cells remain together in a single mass and do not invade or disrupt surrounding tissues. Malignant tumor: tumor cells invade and disrupt surrounding tissues (and are diagnosed as cancer). Metastasis: spread of malignant tumor cells throughout the body (typically through the blood and lymphatic system). Cell cycle: proliferating cells undergo G1, S, G2 and M phase for cell division, the process is controlled by a set of proto-oncogenes proto-oncogenes and tumor suppressor genes. Differentiation: Cells reduce proliferation and become certain type of cell performing certain function. Differentiation correlates correlates with loss of ability to proliferate. Stem cell: cells divide without undergoing differentiation, and capable of self-renewal.
CANCER DUE TO ACTION OF DNA REPAIR GENES Mutagens or DNA Replication
Proto-oncogene
Malignant Transformation Mutated DNA Repair genes
CELL PROLIFERATION
DNA damages
Repaired DNA
24: Imm unology •
22: The Mechanics of Cell Division •
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Mitosis: Mitosis involves the dividing of the nucleus and the surrounding cell. Mitosis occurs after the successful replication of the cell’s DNA. Prophase: the normally loosely packed chromatin is condensed into the chromosome. The two centrosomes (Mitotic Spindle) begin to extend microtubules, in preparation for attaching to the chromosomes. Metaphase: the chromosomes are aligned at the center of the cell at a region called the metaphase plate. Each of the sister chromatids (2 per chromosome) are attached to opposite poles of the mitotic spindle. Anaphase: There are many proteins that make up the anaphase promoting complex. This complex plays a key r ole in allowing the cell to successfully separate the sister chromatids in the 2 future daughter cells. Cytokinesis: Eventually, as cytokinesis progresses, the cleavage furrow deepens until the plasma membrane and its contents are divided.
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Immune System: It is a unique adaptive defensive system that has evolved in vertebrates to protect them from invading pathogenic microorganisms and cancer. Immunity: All those physiological mechanisms that endow the animal with the capacity to recognize materials as foreign to itself and to neutralize eliminate or metabolize them with or without injury to its own tissues. Antigen: Any substance (usually foreign) that bind specifically to an antibody or a T cell receptor. Antibody: A protein (immunoglobulin) that recognizes a particular antigen and binds specifically to it. Innate immunity or native immunity: It is the resistance to infections, which an individual possesses by virtue of his genetic and constitutional make-up. make-up. It is not affected by prior contact with microorganisms or immunization. Adaptive immunity or acquired immunity: It is the resistance that an individual acquires during life, as distinct from the inborn innate immunity. Adaptive immune responses exhibit four immunological attributes: Specificity, Diversity, Memory, Self and non-self recognition. Protect against Bone Bone marrow marrow Thymus Thymus
Lymphatic Lymphatic system systemof of ducts ducts&&nodes nodes
Spleen Spleen
Immune Immune System System
Pathogens Pathogens Bacteria Bacteria
Viruses Viruses Multi-cellular Multi-cellular
Protozoa Protozoa
parasites parasites
Fungi Fungi Leucocytes Leucocytes
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