BIOL 3450 Exam 1

serine proteases demonstrate how enzymes active site works

~(1) substrate peptide and enzyme binding site form H bonds that resemble β sheet (2) key side chain of substrate determines which peptide in substrate is to be cleaved [and extends this into enzymes side chain specificity binding pocket where at the bottom there is negatively charged side chain of enzymes Asp-189] ~trypsin has preference for substrates w/ long positively charged side chains, chymotrypsin prefers large aromatic groups and elastase prefers small side chains of Gly and Ala ~in catalytic site all 3 enzymes use hydroxyl group on side chain of serine to catalyze hydrolysis of peptide bonds in protein substrates [catalytic triad of Ser-195, His-57, Asp-102 cooperates in breaking peptide bond which initially forms unstable transition state w/ 4 groups attached to C] ~breaking C-N peptide bond releases part of substrate (NH₃-P₂) while other part remains covalently attached to enzyme via ester bond to serines O [replacement of this O by one from water leads to release of final product (P₁-COOH) [tetrahedral intermediate transition states are partially stabilized by H bonding from enzymes oxyanion hole]

centrifugation can separate particles and molecules that differ in mass or density

~2 particles in suspension (cells, cell fragments, organelles, molecules) w/ diff masses or densities will settle to the bottom of a tube at diff rates ~used for (1) as a preparative technique to separate one type of material from others (2) analytical technique to measure physical properties of macromolecules [sedimentation constant (s) of protein is measure of its sedimentation rate which is expressed in (S)- ex: ribosome is 80S] ~initial step in protein purification from cells is separation of water soluble proteins from insoluble cellular material by differential centrifugation [mechanically broken cells are put in tube and spun so large fragments collect at bottom (pellet) and soluble proteins remain in supernatant] ~water soluble proteins can be separated by centrifugation through solution of increasing density called density gradient [when protein mixture is layered on top of sucrose gradient and subjected to centrifugation each protein migrates down tube at rate controlled by factors that affect sedimentation constant] ~rate zonal centrifugation- proteins start at top of tube and separate into bands of proteins of diff masses [seldom effective in determining precise molecular weights b/c variations in shape also affect sedimentation rates]

chemical building blocks of life

~3 main macromolecules are proteins, nucleic acids and polysaccharides [which are polymers composed of multiple covalently linked monomers] ~proteins are polymers containing 100-1000 amino acids linked by peptide bonds ~nucleic acids are polymers containing hundreds to millions of nucleotides linked by phosphodiester bonds ~polysaccharides are branched polymers of monosaccharides (sugars) linked by glycosidic bonds ~macromolecules can also be assembled using noncovalent interactions- bilayer structure of cellular membrane is built by noncovalent assembly of small molecules called phospholipids

hydrolysis of ATP releases substantial free energy and drives many cellular processes

~ATP is type of usable nergy that cells can spend in order to power their activities [useful energy in ATP is contained in phosphoanhydride bonds which are covalent bonds formed from the condensation of 2 molecules of phosphate by loss of water] ~ATP has 2 key phosphoanhydride bonds (also called phosphodiester) [when these bonds are hydrolyzed the energy is releases with a ∆G=-7.3 kcal/mol] ~reason breaking this bond gives so much energy is b/c ADP and Pi are highly charged at neutral pH so when these charges are broken apart from each other lots of energy is released ~energy coupling- some of the energy stored in phosphoanhydride bond is transferred to one of the reactants by breaking bond in ATP and forming a covalent bond b/w released phosphate group and one of the reactants ~ex for B+C→D B + Ap~p~p → B~p + Ap~p B~p + C → D + Pi so overall reaction is B + C + ATP ↔ D + ADP + Pi

noncovalent binding of calcium and GTP are widely used as allosteric switches to control protein activity

~Ca²⁺/calmodulin mediation switching- concentration of free Ca²⁺ in cytosol is kept very low by membrane transport proteins that continually pump excess Ca²⁺ out of cytosol [Ca²⁺ permeable channels in cell surface membranes open and allow extracellular Ca²⁺ to flow into cell- this rise is sensed by Ca²⁺ binding proteins which alter cellular behavior by turning activities of other proteins on and off] [Ca²⁺ binding proteins bind Ca²⁺ using EF hand structural motif (calmodulin)] [calmodulin contains 4 Ca²⁺ binding EF hands and binding of Ca²⁺ to calmodulin causes conformational change that permits calmodulin to bind to conserved sequences in target proteins thereby switching activities on/off] ~guanine-nucleotide binding proteins- all GTPase switch proteins exist in 2 forms an active form w/ bound GTP that can influence activity of specific target proteins to which they bind and inactive form w/ bound GDP [switch is turned on when GTP replaces GDP and conformation changes] [switch is turned off when slow GTPase activity of protein hydrolyzes GTP converting it to GDP and leading to conformation change] [cells contain variety of proteins that modulate baseline rate of GTPase activity for any GTPase switch and so control how long the switch remains on]

native DNA is double helix of complementary antiparallel strands

~DNA consists of 2 associated polynucleotide strands that wind together to form double helix [2 sugar-phosphate backbones are on outside of double helix and bases project into interior] ~orientation of strands in antiparallel so 5' to 3' directions are opposite [strands are held together by formation of base pairs b/w strands (A is paired w/ T through 2 H bonds and G is paired w/ C through 3 H bonds)] ~presence of thousands of H bonds in DNA molecule contribute to stability of double helix [hydrophobic and van der waals interactions b/w stacked base pairs further stabilizes structure] ~most DNA in cells is righthanded helix (B form DNA) [helix makes complete turn every 3.6 nm and so there are 10-10.5 base pairs per turn] [on outside of N form DNA spaces b/w strands form 2 helical grooves the major and minor grooves] [atoms on edge of each base within grooves are accessible from outside helix forming 2 types of binding surfaces] ~A form DNA is wider and shorter w/ deeper major groove and more narrow and shallow minor groove ~unlike α helices in proteins there are no H bonds parallel to axis of DNA helix [this allows DNA to bend which is important for dense packing of DNA in chromatin]

hydrolysis of ATP releases substantial free energy and drives many cellular processes (continued)

~alternative mechanism of energy coupling is to use the energy released by ATP hydrolysis to change conformation of molecule to energy rich stressed state [energy stored as conformational stress can be released as molecule relaxes back into its unstressed conformation] ~transport of molecules into and out of cell has positive ∆G and requires input of energy [in this case ∆G=RTln[Cin]/[Cout] where [Cin] is initial concentration of substance inside cell and [Cout is concentration outside cell]

duplex DNA is unwound and daughter strands are formed at DNA replication fork

~DNA polymerase cant initiate chain synthesis but it needs a short preexisting RNA strand called a primer to begin chain growth [w/ primer base paired to template strand DNA polymerase adds dNTPs to free hydroxyl group at 3' end of primer] ~unwinding of parental DNA strands is by helicase and begins at unique segments in DNA called replication origins ~once helicase has unwound parental DNA at origin specialized RNA polymerase called primase forms RNA primer [this primer is then elongated by DNA polymerase forming new daughter strand] ~replication fork- DNA region where all proteins carry out synthesis of daughter strand [replication fork moves as synthesis goes on and local unwinding of DNA produces torsional stress which is released by topoisomerase] ~synthesis of leading strand can proceed continuously from RNA primer in 5' to 3' direction (the same movement as the replication fork) ~b/c growth of lagging strand must occur in 5' to 3' direction a new primer must be synthesized every hundred bases [each of these primers is elongated in 5' to 3' direction forming discontinuous segments called Okazaki fragments- RNA primer of each Okazaki fragment is removed and replaced by DNA chain growth from neighboring fragment] [DNA ligase joins adjacent fragments]

protein primary structure can be determined by chemical methods and from gene sequences

~Edman degradation- free amino group of N terminal is labeled and then cleaved from polypeptide and identified by high pressure liquid chromatography [polypeptide is left one residue shorter w/ new amino acid at N terminus and cycle is repeated until all residues have been identified] ~peptide mass fingerprint- list of molecular weights of peptides that are generated from protein by digestion w/ specific protease [molecular weights of parent protein and its fragments are used to search genome databases for similar size proteins w/ identical or similar mass maps

chaperonins

~Hsp60s are cylindrical supramolecular assemblies [there are 2 distinct groups of chaperonins that differ in their structure, detailed molecular mechanisms and locations] ~group I chaperonins (found in prokaryotes, chloroplasts and mitochondria) are composed of 2 rings each having 7 subunits that interact w/ a homoheptameric co chaperone "lid" ~group II chaperonins (found in eukaryotic cells and in archaea) can have 8 to 9 either homomeric and heteromeric subunits in each ring and "lid" function is incorporated in those subunits themselves no separate lid is needed [ATP hydrolysis triggers closing of lid of group II chaperonins] ~GroEL ATPase cycle- (1) partly folded or misfolded polypeptide is captured by hydrophobic residues near entrance of GroEL chamber and enters one of the folding chambers [the second chamber is blocked by GroES lid] [each of the subunits of GroEL can bind ATP and hydrolyze it and release ADP and lead to conformational changes- these changes both control binding of lid and environment inside] (2) polypeptide remains encased in chamber capped by lid where it can undergo folding until ATP hydrolysis induces binding of ATP and diff GroES to other ring- this causes GroES lid and ADP bound to peptide containing ring to be released opening chamber and polypeptide diffuses out ~capping of one chamber by GroES to permit sequestered substrate folding in that chamber is accompanied by release of substrate from the chamber of the second ring

molecular chaperones

~Hsp70 and its homologs are the major chaperones in all organisms [when bound to ATP, Hsp70 assumes open form to which exposed hydrophobic substrate binding pocket binds to exposed hydrophobic regions of incompletely folded or partially denatured target protein] ~hydrolysis of bound ATP causes molecular chaperone to assume closed form that binds its substrate protein more tightly and facilitates protein folding [exchange of ATP for ADP causes conformational change in chaperone that releases target protein] [if target is now properly folded it cant rebind to Hsp70 but if it remains partially unfolded it can bind again] ~another molecular chaperone is Hsp90 (not in archaea) which helps cells cope w/ denatured proteins generated by stress and ensures some of their substrates can be converted from inactive to active state or otherwise held in functional conformation ~for Hsp90 clients bind to open conformation, ATP binding leads to interaction of ATP binding domains and formation of closed conformation, hydrolysis of ATP plays important role in activating client proteins and their release from Hsp90

proteolytic cleavage irreversibly activates or inactivates some proteins

~activation or inactivation of protein function by proteolytic cleavage is irreversible mechanism for regulating protein activity ~elaborate protease cascades (one protease activating inactive precursors of others) that can amplify an initial signal play important roles in several systems such as blood clotting cascade ~protein self splicing- internal segment of polypeptide is removed and ends of polypeptide are ligated [autocatalytic process that proceeds by itself w/o participation of other enzymes] ~compartmentation of proteins in parts of the cell provides opportunity for controlling delivery of substrate or exit of product and also permits reactions to take pace simultaneously in different parts of the cell

amino acids differing only in their side chains compose proteins

~all amino acids have characteristic structure consisting of central alpha carbon atom (Cα) bonded to 4 diff groups: an amino group (NH₂), a carboxyl group (COOH), a H atom and the side chain/R group ~α carbon in all amino acids except glycine is asymmetric and exist as D and L isomers [these 2 isomers cant be interconverted w/o breaking and reforming chemical bond] [only L form of amino acids are found in proteins] ~amino acids w/ nonpolar side chains are hydrophobic [side chains of alanine, valine, leucine and isoleucine are branched hydrocarbons that dont form a ring and are nonpolar so are called alipathic amino acids] [methionine is also nonpolar and contains one sulfur] [phenylalanine, tyrosine and tryptophan have hydrophobic aromatic rings in side chain] ~amino acids w/ polar side chains are hydrophilic [side chains are normally charged] [arginine and lysine have positively charged side chains and are called basic amino acids] [aspartic acid and glutamic acid have negatively charged side chains and are called acidic amino acid] [histidine has side chain w/ ring w/ 2 nitrogens which can shift from positive charge to uncharged depending on environment]

phospholipids are conserved building blocks of all cellular membranes

~all cellular membranes are composed primarily of bilayer (2 layers) of phospholipid molecules ~these molecules have hydrophilic end and hydrophobic end where hydrophilic ends are directed towards surface and hydrophobic ends are buried within interior [impermeable to water, all ions and virtually all hydrophilic small molecules] ~membrane contains groups of proteins that allow specific ions and small molecules to cross [other membrane proteins serve to attach cell to other cells or to polymers that surround it [and others give cell its shape and allow its shape to change] ~membranes are made by incorporation of lipids and proteins into existing membranes in parental cell and these are divided b/w daughter cells by fission

alternative splicing increases number of proteins expressed from single eukaryotic gene

~alternative splicing- presence of multiple introns permits expression of multiple related proteins from a single gene [alternative splicing is important mechanism for production of diff forms of proteins (isoforms) by diff types of cells]

amino acids differing only in their side chains compose proteins (continued)

~asparagine and glutamine are uncharged but have polar side chains w/ amide groups that H bond ~serine and threonine are uncharged but have polar hydroxyl groups which H bond ~cysteine has reactive sulfhydryl group (-SH) that turns into thiolate anion (S⁻) [thiolate anion plays important role in catalysis of enzymes that destroy proteins] [when 2 cysteines come together the sulfhydryl groups are oxidized each releasing a proton to form covalent disulfide bond] ~cysteine stabilizes folded structure of some proteins, glycine can fit into tight spaces, proline is rigid and amino group is not available for H bonding [presence of proline creates fixed kink in polymer chain limiting how it can fold] ~there are common modifications to amino acids after they are incorporated into protein- addition of acetyl group (CH₃CO), addition of phosphate (PO₄) to hydroxyl groups in serine, threonine and tyrosine, glycosylation (attachment of linear and branched carbohydrate chains) to asparagine, serine and threonine [reversed by modification of N-acetylglucosamine], hydroxylation of proline and lysine in collagen, methylation of histidine in membrane receptors, γ carboxylation of glutamate in blood clotting factors ~acetylation (addition of acetyl group) to amino group of N terminal is most common chemical modification [controls life span of proteins within cells b/c nonacetylated proteins are rapidly degraded]

non covalent interactions (continued)

~b/c nonpolar molecules dont have charge groups, dipole moments and cant become hydrated they are insoluble in water and are hydrophobic ~hydrocarbons and molecules w/ long hydrocarbon chains are insoluble ~hydrophobic effect- b/c water molecules cant form H bonds w/ nonpolar substances they form cages of rigid H-bonded pentagons and hexagons around nonpolar molecules [this state is energetically unfavorable b/c it decreases entropy (randomness) of water molecules] ~if nonpolar molecules aggregate w/ hydrophobic ends facing each other entropy increases relative to unaggregated state [so this is why nonpolar molecules aggregate together in water] ~packaging of lipids (nonpolar molecules) into special carriers called lipoproteins permits their efficient transport in blood (aqueous) b/c of hydrophobic effect ~complementary shapes/charges/polarity/hydrophobicity of 2 protein surfaces permit multiple weak interactions which in combination produce strong interaction and tight binding [particular surface of molecule can bind tightly only to one or very limited number of molecules]

planar peptide bonds limit shapes into which proteins can fold

~b/c peptide bond behaves partially like a double bond the carbonyl C and amide N and atoms directly bonded to those have to lie in a fixed plane ~only flexibility in polypeptide chain backbone is rotation of fixed planes of adjacent peptide bonds w/ respect to one another about 2 bonds: the α carbon-amino N bond (phi angle) and the α carbon-carbonyl C bond (psi angle) [only a limited number of these angles are possible b/c side chain atoms would come to close to one another]

genomics, cell architecture and cell function

~bacterial cells consist of single closed compartment containing the cytoplasm and bounded by the plasma membrane [although they dont have a defined nucleus the single circular DNA genome is extensively folded and condensed into central region of cell] ~some bacteria also have a type of cell membrane called a mesosome which is associated with synthesis of DNA and secretion of proteins ~bacterial cells posses a cell wall which lies adjacent to external side of plasma membrane [cell wall is made of layers of peptidoglycan] [some bacteria have thin inner cell wall and outer membrane separated from inner cell wall by periplasmic space- these bacteria are gram negative] ~gram positive bacteria have thicker cell wall and no outer membrane

torsional stress in DNA is relieved by enzymes

~bacterial, viral and mitochondrial DNA are circular [each of 2 strands in circular DNA forms closed structure w/o free ends] [localized unwinding of circular DNA molecule which occurs during DNA replication induces torsional stress into remaining portion of molecule b/c ends arent free to rotate- as a result DNA molecule twists back on itself forming supercoils] ~topoisomerase I- relieves torsional stress that develops in DNA molecules during replication [binds to DNA at random sites and breaks phosphodiester bond in one strand (nick) and broken end winds around uncut strand leading to loss of supercoils and then ligates 2 ends of broken strand] ~topoisomerase II- makes breaks in both strands of double stranded DNA and ligates them [can both relieve torsional stress and link together 2 circular DNA molecules as in the links of a chain]

dissociation constants of binding reactions reflect affinity of interacting molecules

~binding "reactions" involve making and breaking of various noncovalent interactions rather than covalent bonds [ex: binding of ligand to its receptor on surface of cell which triggers intracellular signaling pathway] ~extent to which protein (P) is bound to DNA (D) forming a protein DNA complex (PD) ~P+D→←PD ~dissociation constant (Kd)=[P][D]/[PD] ~the lower the Kd the tighter the binding (higher affinity) of P for D ~sometimes binding of a molecule at one site on a macromolecule can change the 3D shape of a distant site thus altering binding interactions of that distant site w/ some other molecule

chemical reaction is in equilibrium when rates of forward and reverse reactions are equal

~both extent to which reactions can proceed and rate at which they take place determine chemical compositions of cells ~when reactants first mix together (before any product can be formed) the rate of forward reaction depends on initial concentrations ~as more products accumulate forward rate slows (as reactants are reformed) and reverse rate increases [eventually rates equal and system is then at chemical equilibrium] ~Keq- ratio of concentrations of products to reactants when they reach equilibrium [fixed value] ~rate of chemical reaction can be increased by a catalyst but a catalyst doesnt change the Keq

regulating protein function

~catalytic activity of enzymes is regulated so that amount of reaction product is just sufficient to meet needs of cell [steady state concentrations of substrates and products will vary depending on cellular conditions] ~ways to regulate protein activity- (1) cells can increase/decrease steady state level of protein by altering its rate of synthesis, rate of degradation or both (2) cells can change intrinsic activity [ex: change of affinity of substrate binding or fraction of time protein is in active vs inactive conformation] (3) change in location or concentration within the cell/target of proteins activity

molecules of life

~cells acquire small molecules by importing them into the cell or synthesizing them within the cell ~ions, water, sugars, vitamins, amino acids, ATP, hormones, growth factors ~ability to catalyze reactions with one stereoisomer instead of the other ~ATP (which stores readily available chemical energy in 2 of its chemical bonds) can form ADP when one of the 2 bonds is broken and release energy which can be harnessed to power energy requiring processes ~hormones act as signals that direct activities of cells and nerve cells communicate w/ each other by releasing and sensing certain small molecules ~monomers (small molecules) can be joined to form polymers/macromolecules through repetition of a single type of covalent chemical linkage reaction ~cells produce 3 types of large macromolecules- polysaccharides, proteins and nucleic acids ~cell is careful to provide appropriate mix of small molecules needed as precursors for synthesis of macromolecules ~humans have about 10^14 cells and the typical cell size is 10 µm (mass is 1 nanogram) [more microorganism cells in our body then actual human cells] ~cells discovered by Robert Hooke who named them after monks cells ~abiogenesis is the theory of the origin of life while evolution is the theory of how life changes once it exists ~omics- systems biology [genomics- gene (46 DNA molecules comprised of 3x10^9 nucleotide pairs) (human genome is estimated to have 20,000-25,0000 genes), transcriptomics- mRNA, proteomics- protein, metabolomics- structure/function]

life depends on coupling of unfavorable chemical reactions

~cells can carry out energy requiting (endergonic) reactions by coupling them w/ energy releasing (exergonic) reactions if the sum of the 2 reactions has overall net negative ∆G A↔B+X ∆G=+5 kcal/mol X↔Y+Z ∆G=-10 kcal/mol ____________________________ A↔B+Y+Z ∆G°=-5 kcal/mol

ubiquitin marks cytosolic proteins for degradation in proteasomes

~cells mark proteins that should be degraded by covalently attaching to them a linear chain of polypeptide called ubiquitin ~(1) activation of ubiquitin-activating enzyme (E1) by addition of ubiquitin molecule [reaction that requires ATP] (2) transfer of ubiquitin molecule to cysteine residue in ubiquitin-conjugating enzyme (E2) (3) formation of covalent bond b/w carboxyl group of glycine 76 of ubiquitin bound to E2 and amino group of lysine residue in target protein [reaction catalyzed by ubiquitin-protein ligase (E3)] (4) subsequent reactions covalently attach glycine of additional ubiquitin via isopeptide bond to side chain of lysine of previously added ubiquitin to create polyubiquitin chain attached to target protein (5) 19S cap of proteasome recognizes ubiquitin labeled proteins and unfold and transports them into proteasome for degradation (6) Dubs hydrolyze bonds b/w individual ubiquitins recycling ubiquitins for additional rounds of protein mofications

folding of proteins in vivo is promoted by chaperones

~cells require faster more efficient mechanism for folding proteins than sequence alone provides [w/o help cells waste energy in the synthesis of improperly folded or nonfunctional proteins which will have to be destroyed to prevent disrupting cell function] ~chaperones- set of proteins that facilitate proper folding of nascent proteins ~chaperones use ATP binding, ATP hydrolysis to ADP and exchange of new ATP for ADP to induce series of conformational changes essential for their function ~chaperones can fold newly made proteins into functional conformations, disassemble potentially toxic misassembled proteins, assemble and dissemble large multiprotein complexes ~ATP dependent conformational switch is used (1) to optimize folding after one substrate is folded (2) return chaperone to initial state so its available to help fold another molecule (3) set time permitted for refolding ~molecular chaperones- bind to short segment of protein substrate and stabilize unfolded or partly folded protein preventing these proteins from aggregating and being degraded ~chaperonins- form small folding chambers into which unfolded proteins can be sequestered (giving time and appropriate environment to fold properly) ~unfolded and partly folded proteins tend to aggregate into large water insoluble masses from which its difficult for protein to dissociate and then fold into proper conformation

highly specific enzyme and antibody assays can detect individual proteins

~chromogenic and light emitting enzyme reactions- enzymatic activity assays are based on ability to detect loss of substrate or formation of product [b/c of specificity of enzyme for its substrate only samples that contain enzyme will change color in presence of chromogenic substrate- rate of reaction provides measure of quantity of enzyme present] ~antibody assays- presence of antigen that contains epitope can be visualized by labeling antibody w/ enzyme, fluorescent molecule or radioactive isotopes [after antibody binds to protein of interest (antigen) and unbound antibody is washed away substrates of linked enzyme are added and appearance of color or light is monitored- intensity is proportional to amount of antigen in sample] [absence of antibody binding doesnt mean antigen is not present in sample only that epitope is not accessible for binding] ~detecting proteins in gel- either label/stain proteins while they are still within gel or electrophoretically transfer proteins to membrane made of nitrocellulose and detect them ~immunoblotting- combines power of gel electrophoresis and specificity of antibodies [use 2 antibodies- one that is specific for desired protein and one that binds to first and is linked to enzyme that permits detection of first antibody]

monosaccharides covalently assemble into linear and branched polysaccharides (continued)

~common disaccharides are lactose (composed of galactose and glucose) and sucrose (composed of glucose and fructose) ~polysaccharides can function as storage for glucose, as structural components or as adhesives that hold cells together in tissues ~most common storage carb in animals is glycogen (branched polymer of glucose) and in plants is starch (glucose polymer that can occur in unbranched form [amylose] or lightly branched form [amylopectin]) [both of these are made of α glucose] ~cellulose (which gives structure in plant cells) is unbranched polymer of β glucose [human digestive enzymes can digest α glycosidic bonds in starch but not β glycosidic bonds in cellulose] ~peptidoglycan- polysaccharide chain cross linked by peptide cross bridges that makes up bacterial cell wall ~enzymes that make glycosidic bonds are specific for α or β anomer of sugars ~any 2 sugar molecules can be linked in variety of ways b/c monosaccharides have multiple hydroxyl groups that can participate in formation of glycosidic bonds [also one monosaccharide has potential to be linked to more than 2 other monosaccharides creating a branching point and nonlinear polymers] ~modifications to sugars include addition of a phosphate or nucleotide [epimerase enzyme that interconverts diff monosaccharides often does using nucleotide sugars rather than unsubstituted sugars]

nucleic acids carry coded information for making proteins at right time and place

~complementary matching of the 2 strands of DNA is so strong that if complementary strands are separated they will spontaneously zip back together under right salt concentrations and temp conditions ~nucleic acid hybridization is extremely useful for detecting one strand by using the other ~genes contain 2 parts- coding region specifies amino acid sequence of protein and regulatory region binds specific proteins and controls when and in which cells the protein is made ~cells convert coded info in DNA into proteins by transcription (coding region of gene is copied into single stranded RNA whose sequence is the same as one of the strands of the DNA) (RNA polymerase catalyzes linkage of nucleotides into RNA chain using DNA template) ~translation is where ribosome (composed of RNA and protein) assembles and links together amino acids in precise order dictated by mRNA sequence according to genetic code ~ribosome is built of 4 RNA chains that bind to more than 50 proteins to make precise and efficient mRNA reader and protein synthesizer ~most chemical reactions are catalyzed by proteins but the formation of peptide bonds that connect amino acids is catalyzed by RNA molecules ~small miRNAs bind to and repress activity of target mRNAs (long noncoding RNAs bind to DNA or chromosomal proteins and so affect chromosome structure and RNA synthesis/processing/stability ~transcription factors- bind to specific sequences of DNA and act as switches either activating or repressing transcription of particular genes

protein structure and function

~conformations- linear polymer of protein will fold into only one or a few closely related 3D shapes [conformation plus chemical properties of distinct side chains determine protein function] ~structural proteins (determine shapes of cells and direct intracellular movement of molecules and organelles), scaffold proteins (bring other proteins together into ordered arrays to perform specific functions more efficiently than if those proteins were not assembled together), enzymes (catalyze chemical reactions), membrane transport proteins (permit flow of ions and molecules across membranes), regulatory proteins (acts as signals/sensors/switches to control the activities of cells by altering functions of other proteins and genes), motor proteins (move other proteins/cells) ~molecular machines- large complexes that some proteins assemble into to accomplish diverse tasks ~proteins perform diverse functions by binding to other macromolecules and small molecules which induces conformational change in protein and influences activity ~catalysis- appropriate folding will place some amino acid side chains and carboxyl/amino groups of its backbone into positions that permit the catalysis of covalent bond rearrangements

viruses are cellular parasites that are employed in biology research

~conserved proteins comprise structural proteins that form parts of the mature virus particle (virion) and proteins that catalyze steps in viral DNA replication [late in human infection human cells produce about half of the RNAs synthesized are viral and most proteins produced are viral] ~many methods for genetically manipulating cells depend on using viruses to convey DNA molecules into cells] ~altered viruses (vectors) where genetic material that is potentially harmful is replaced w/ other genetic material (including human genes) can enter cells w/ introduced genes ~diseases caused by defective genes may be treated using viral vectors to introduce normal copy of defective gene into patients [gene therapy]

ATP is generated during photosynthesis and respiration

~constantly replenishing ATP requires that cells continuously obtain energy from environment [for most cells ultimate source of energy used to make ATP is sunlight] ~process of photosynthesis uses sunlight to synthesize ATP from ADP and Pi [ATP produced in photosynthesis is hydrolyzed to provide energy for conversion of carbon dioxide to 6 sugar carbons (carbon fixation)] ~6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂ + energy ~free energy in sugars and other molecules derived from food is released in glycolysis and cellular respiration [during cellular respiration energy rich molecules in food are oxidized to carbon dioxide and water] ~C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O ~oxidation of 1 molecule of glucose can synthesize as many as 30 molecules of ATP from 30 molecules of ADP ~catabolism of glucose is major pathway for generating ATP in non plant cells (catabolism of fatty acids can also be source of ATP)

enzymes active site binds substrates and carries out catalysis

~critically important amino acids (which usually come from diff parts of the linear sequence) are brought into proximity forming cleft in surface called active site ~active site is small part of protein while the rest is involved in folding, regulation of active site and interactions w/ other molecules ~active site has 2 functionally important regions- substrate binding site [recognizes and binds substrate] and catalytic site [carries out chemical reaction once substrate is bound] ~specificity of enzymes for substrates is consequence of molecular complementarity ~rate of enzymatic reaction was proportional to substrate concentration at low substrate concentrations but as substrate concentration increased the rate reached a maximal velocity (Vmax) and became substrate concentration independent w/ value of Vmax being directly proportional to amount of enzyme present in reaction mixture ~rate V₀ of formation of product at particular substrate concentration [S] is given by V₀=Vmax [S]/[S]+Km where Km (Michaelis constant) is a measure of affinity of enzyme for its substrate [the smaller the value of Km the more effective the enzyme is and the lower the substrate concentration needed to reach half maximal velocity] ~intracellular concentration of substrate is approximately the same as (or a little greater than) the Km of the enzyme to which it binds ~turnover number- max number of substrate molecules converted to product at single enzyme active site per second ~many enzymes catalyze conversion of substrates to products by dividing process into multiple, discrete chemical reactions that involve multiple enzyme substrate complexes [energy profiles for multistep reactions involve multiple hills and valleys]

biological fluids have characteristic pH values

~cytosol of cells normally has pH of 7.2 but interior of certain cells (lysosomes) have lower pH ~in neutral solutions many amino acids exist in doubly ionized form (where carboxyl group has lost proton and amino group has accepted one) [at extreme pH values only one of these 2 ionizable groups will be charged] ~pH=pKa+log[A⁻]/[HA] where pKa=-logKa ~cells have reservoir of weak bases and weak acids (buffers) to make sure cytoplasmic DNA remains relatively constant despite small fluctuations in amount of H₃O⁺ and OH⁻ being generated by metabolism ~buffering capacity is the ability of a buffer to minimize changes in pH [depends on concentration of buffer and relationship b/w pKa value and pH] ~phosphoric acid is important factor in buffering pH of cytoplasm (because it can lose 3 protons)

regulated synthesis and degradation of proteins is fundamental property of cells

~degradation removes proteins that are potentially toxic/improperly folded/damaged (including products of mutated genes and proteins damaged by metabolites or stress) ~controlled destruction of otherwise normal proteins provides powerful mechanism for maintaining appropriate levels of proteins and their activities and for permitting rapid changes in these levels to help cells respond to changing conditions ~pathways for degradation- enzymes within lysosomes [lysosomal degradation is directed primarily towards aged or defective organelles of cell and toward extracellular proteins taken up by cell] and cytoplasmic protein degradation by proteasomes

domains are modules of tertiary structure

~domains- distinct regions of protein structure ~functional domain- region of protein that exhibits a particular activity characteristic of the protein [functional domains are identified by whittling down protein to smallest active fragment w/ aid of proteases] [have activity independent from the rest of the protein] ~structural domain- region of 40 or more amino acids in length arranged in a single stable and distinct structure comprising one or more secondary structures [can fold into characteristic structures independently of the rest of the protein in which they are embedded] [distinct structural domains can be linked together to form large multidomain protein] [large proteins tend to be mosaics of diff domains that confer distinct activities and this can perform different functions simultaneously] [there are about 1000 diff types of structural domains in all proteins] ~topological domains- regions of proteins that are defined by their distinctive spatial relationships to the rest of the protein [each of these can comprise one or more structural and functional domains] [ex: proteins associated w/ cell surface membranes]

genetic diseases elucidate important aspects of cell function

~duchenne muscular dystrophy (DMD) is X chromosome linked disorder that results in cardiac or respitory failure in men during their 30s [people w/ DMD carry mutation in gene encoding protein dystrophin] [this protein binds to actin filaments that are part of cytoskeleton and to complex of muscle plasma membrane proteins (sarcoglycan complex)] ~mutations in dystrophin can disrupt link b/w exterior and interior of muscle cells and cause muscle weakness and eventual death

DNA can undergo reversible strand separation

~during replication and transcription the strands of double helix must separate to allow internal edges of bases to pair w/ bases of nucleotide being polymerized ~denaturation of DNA- as thermal energy increases the resulting increase in molecular motion breaks H bonds and strands separate driven apart by electrostatic repulsion of negatively charged backbone of each strand ~melting temp (Tm) at which DNA separates depends on several factors- (1) molecules w/ greater proportion of GC pairs require higher temp to denature b/c of 3 bonds (2) ion concentration b/c when ion concentration is low it reduces Tm (3) agents that destabilize H bonds lower Tm (4) extreme pH lowers Tm ~single stranded DNA molecules that result from denaturation form random coils w/o organized structure [lowering temp, increasing ion concentration or neutralizing pH causes renaturation of strands- renaturation is dependent on time, DNA concentration and ionic concentration]

translation is terminated by release factors when stop codon is reached

~eRF1 (whose shape is similar to tRNA) acts by binding to A site and recognizing stop codons directly [eRF3 bound to GTP works w/ eRF1 to promote cleavage of tRNA releasing completed protein chain] ~release of completed protein leaves free tRNA in P site and mRNA still associated w/ 80S ribosome w/ eRF1 and eRF3-GDP bound in A site ~ribosome recycling occurs when post termination complex is bound by protein ABCE1 which uses energy from ATP to separate subunits and release mRNA ~eIF1, eIF1A and eIF3 are also required and load into 40S subunit making it ready for another round of initiation

amino acids become activated when covalently linked to tRNAs

~each of the 20 different synthetases recognize one amino acid and all its compatible tRNAs [energy of bond b/w tRNA and amino acid drives formation of peptide bonds linking adjacent amino acids in growing polypeptide chain] ~aminoacyl-tRNA synthetases recognize tRNAs by interacting w/ anticodon loop and acceptor stem [can sometimes make mistakes and link with wrong tRNA but enzyme has proof reading ability that checks fit in their amino acid binding pocket] ~if wrong amino acid becomes attached the bound synthetase catalyzes removal of amino acid from tRNA

body plan and rudimentary tissues form early in embryonic development

~early stages of development is characterized by rapid cell division and differentiation of cells into tissues ~embryonic body plan (the spatial pattern of tissues and body parts) emerges from 2 influences- program of genes that specify pattern of body and local cell interactions that induce diff parts of the program ~protostomes develop mouth close to transient opening in early embryo (blastopore) and have ventral nerve cord [worms/insects/mollusks] and deuterostomes develop anus close to transient opening and have dorsal central nervous system [echinoderms/vertebrates] ~patterning genes specify general organization of organism (beginning w/ major body axes and ending w/ body segments such as head/tail/chest) ~conservation of axial symmetry is explained by conserved patterning genes [conservation of body plan reflects evolutionary pressure to preserve commonalities in molecular and cellular mechanisms controlling development in diff organisms]

electrophoresis separates molecules on basis of their charge to mass ratio

~electrophoresis is technique for separating molecules in mixture under influence of applied electric field [dissolved molecules in electric field move at speed determined by their charge to mass ratio] [if molecules have same mass and shape the one w/ greater net charge will move faster toward electrode of opposite polarity] ~when mixture of proteins is placed in gel smaller proteins migrate faster through gel b/c gel acts as sieve (smaller species able to go through pores faster) [long asymmetric molecules migrate more slowly than spherical ones of same mass] ~SDS treatment eliminates effects of differences in shape in native structures so chain length (mass) is principal element determining migration rates ~in two dimensional gel electrophoresis proteins are separated sequentially first by charges and then by masses [this is for proteins have very similar masses] ~isoelectric focusing (IEF)- gel has pH gradient and charged protein is placed on one end and migrates until it reaches its isoelectric point (pI) which is the pH at which the net charge of the protein is 0 [now that it has no net charge it will not migrate any farther]

all eukaryotic cells have many of the same organelles and other subcellular structures

~eukaryotic cells contain extensive internal membranes that enclose organelles and separate them from the rest of the cytoplasm ~many organelles are surrounded by single phospholipid membrane but nucleus, mitochondria and chloroplast are surrounded by 2 membranes ~lysosomes are organelles filled with degradative enzymes that break down worn out or obsolete parts into small molecules that can be discarded or recycled [interior of lysosome is much more acidic than surrounding cytosol which aids in break down of materials] [to create low pH environment proteins located in lysosomal membrane pump H ions into lysosome using energy from ATP] ~peroxisomes are another type of small organelle found in all eukaryotic cells that is specialized for breaking down lipid components of membranes ~3 classes of fibers compose cytoskeleton- mictrotubules (built of polymers of protein tubulin), microfilaments (built of protein actin) and intermediate filaments (built of one or more rod shaped protein subunits) ~cytoskeleton gives cell strength and rigidity to help maintain cell shape [fibers also control movement of structures within cell which provides tracks along which organelles move] ~some proteins are made on ribosomes that are free in cytoplasm and are moved into nucleus or directed to mitochondria, etc depending on function [proteins to be secreted from cell are made on ribosomes associated with the ER] [protein chains produced on ER move to golgi complex where they are further modified before being forwarded to final destination- these proteins contain sequences of amino acids that serve as addresses]

multicellularity requires cell cell and cell matrix adhesions

~evolution of multicellular organisms most likely began when cells remained associated in small colonies after division instead of separating into individual cells ~multicellularity occurred in eukaryotes whose cells became differentiated and organized into tissues (where cells performed specialized common function) ~animal cells are often "glued" together into a chain/ball/sheet by cell adhesion proteins (CAMs) [some CAMs bind cells to one another or bind cells to extracellular matrix] [matrix cushions cells and allows nutrients to diffuse toward them and waste products to diffuse away] ~basal lamina- specialized tough matrix comprised of collagen forms supporting layer underlying cell sheets and prevents them from ripping apart ~plasmodesmata- cytoplasmic bridges that connect interlocking cell walls surrounding cells in plants

change in free energy determines if a chemical reaction will occur spontaneously

~exergonic- releases energy [products contain less energy than reactants] [take place spontaneously and liberated energy is released as heat] ~endergonic- absorbs energy [products contain more energy than reactants] [if there is no external source of energy to drive endergonic reaction it cant take place] ~chemical reaction occurs spontaneously when free energy of products is lower than free energy of reactants [∆G=Gproducts-Greactants] ~if ∆G is negative forward reaction is spontaneous (exergonic) and if ∆G is positive forward reaction is nonspontaneous (endergonic) ~if ∆G is 0 both forward and reverse reactions occur at equal rates so system is at equilibrium ~in exothermic reaction ∆H is positive (enthalpy) and in endergonic ∆H is positive ~actual change of ∆G during reaction is influenced by temp, pressure, and initial concentrations of reactants and products (also pH of solution if reaction takes place in aqueous solution) ~∆G=∆G°+RTlnQ=∆G°+RTln[products]/[reactants]

nonstandard base pairing often occurs between codons and anticodons

~explanation for smaller number of tRNAs than codons is in capability of single tRNA anticodon to recognize more than one codon corresponding to given amino acid ~wobble position- third base in mRNA codon and corresponding first base in tRNA anticodon ~nonstandard interactions can occur b/w bases in wobble position- ex: GU base pair [which structurally fits almost as well as GC pair] so anticodon w/ G in wobble position can base pair w/ 2 corresponding codons that have either C or U in third position ~many tRNAs have I (deaminated product of adenine) in wobble position b/c it can form nonstandard base pairs w/ A, C and U

members of protein families have common evolutionary ancestor

~family comprises proteins w/ clear evolutionary relationship (>30% identity or additional structural and functional info showing common descent but <30% identity) while a superfamily comprises protein w/ only a probable common evolutionary origin (lower percent sequence identities but one or more common motifs or domains)

equilibrium constant reflects extent of chemical equilibrium

~for aA+bB+cC→←zZ+yY+xX ~Keq=[X]^x[Y]^y[Z]^z/[A]^a[B]^b[C]^c ~rate forward=Kf[A]^a[B]^b[C]^c ~rate reverse=Kr[X]^x[Y]^y[Z]^z ~at equilibrium the forward and reverse rates are equal so rate forward/rate reverse=1 ~Keq=Kf/Kr

∆G° of a reaction can be calculated from its Keq

~for system at equilibrium ∆G°=-2.3RTlogKeq or Keq=10^-(∆G°/2.3RT) ~if ∆G is negative at equilibrium the formation of products is favored and if its positive formation of reactants is favored ~velocity (V) at which products are generated from reactants during reaction under given set of conditions (temp, pressure, reactant concentrations) will depend on concentration of material in transition state which in turn will depend on activation energy and characteristic rate constant (v) at which transition state is converted to products ~the higher the activation energy the lower the fraction of reactants that reach transition state and slower overall rate of reaction ~V=v[reactants]×10^-(∆G‡/2.3RT) ~catalysts accelerate reaction rates by lowering relative energy of transition state and activation energy required to reach it

tissues organized into organs

~function of organ is determined by specific functions of its component tissues and each type of cell in tissue produces the specific groups of proteins that enable tissue to carry out its functions

messenger RNA carries info from DNA in 3 letter genetic code

~genetic code is triplet code w/ codon being read from specified starting point in mRNA ~of the 64 possible codons in genetic code 61 specify individual amino acids and 3 are stop codons [most amino acids are encoded by more than one codon- diff codons for given amino acid are said to be synonymous] ~reading frame- sequence of codons that runs from start codon to stop codon [some mRNAs contain overlapping info that can be translated in different reading frames yielding diff polypeptides]

amino acid sequence of protein determines how it will fold

~if phi and psi angles were limited to only 8 combinations and n-residue long peptide would potentially have 8^n conformations ~native state- any particular protein adopts only one or just a few very closely related conformations called native state [native state is most stably folded form of molecule and one that permits it to function normally] ~denaturation can be induced by thermal energy from heat, extremes of pH that alter charges of side chains, exposure to denaturants which disrupt structure stabilizing noncovalent interactions ~under denaturing conditions a pop of uniformly folded molecules is converted into collection of many unfolded molecules that have many diff non-native and biologically inactive conformations ~info contained in proteins primary structure can be sufficient to direct correct refolding

noncovalent binding permits allosteric (or cooperative) regulation of proteins

~in addition to regulating amount of protein cells can also regulate the intrinsic activity of a protein ~allostery- refers to any change in proteins tertiary or quaternary structure or both induced by noncovalent binding of a ligand ~when ligand binds to one site A in protein and induces conformational change and change in activity of different site B then ligand is called allosteric effector and site A is allosteric binding site and protein is allosteric protein ~allosteric change can either induce an increase or decrease in protein activity [negative allostery often involves end product of pathway binding to and reducing activity of enzyme that catalyzes rate controlling step for that pathway (end product or feedback inhibition)] ~cooperativity- refers to influence (positive or negative) that binding of ligand at one site has on binding of another molecule of same type of ligand at different site [ex: hemoglobin- binding of single ligand (O₂) increases affinity of binding of next oxygen molecule] ~b/c of sigmoidal shape of oxygen saturation curve of hemoglobin it takes only a 4x increase in O concentration for percent saturation of O binding sites in hemoglobin to go from 10% to 90% [if it was noncooperative binding it would take 81x increase] ~cooperativity permits hemoglobin to take up O efficiently in lungs (where O concentration is high) and unload it in tissues (where O concentration is low) ~cooperativity amplifies the sensitivity of a system to concentration changes in its ligands providing a selective evolutionary advantage

cells can transform one type of energy into another

~in photosynthesis radiant energy of light is transformed into chemical potential energy of covalent bonds b/w atoms in sucrose or starch molecule ~in muscles and nerves chemical potential energy stored in covalent bonds is transformed into kinetic energy of muscle contraction or electric energy of nerve transmission ~in all cells potential energy (released by breaking certain chemical bonds) is used to generate potential energy in form of concentration potential gradients

stages in transcription

~initiation- (1) RNA polymerase recognizes specific binding site the promoter (2) after binding RNA polymerase separates DNA strands to make bases in template strand available for pairing (3) RNA polymerase melts 12-14 base pairs (transcription bubble) of DNA around transcription start site [this allows template strand to enter active site of enzyme that catalyzes bond formation b/w rNTPs] (4) initiation is complete when first 2 rNTPs of RNA chain are linked by phosphodiester bond ~elongation- (1) RNA polymerase dissociates from promoter and moves along template one base at a time (opening double stranded DNA in front of its direction of movement ) (2) one ribonucleotide at a time is added to 3' end of growing RNA chain by polymerase [8 nucleotides at 3' end of RNA strand remain base paired to template DNA strand in transcription bubble] [elongation complex (RNA polymerase, template DNA and growing RNA strand) is especially stable] ~termination- (1) completed RNA molecule is released from RNA polymerase and polymerase dissociates from template DNA (2) RNA polymerase is free to transcribe same gene again or another gene

several forms of energy are important in biological systems

~kinetic energy is the energy of movement (motion of molecules) ~potential energy is stored energy (energy stored in covalent bonds) ~for heat to do work it must flow from region of high temp (where average speed of molecular motion is greater) to one of lower temp ~thermal energy is used to maintain constant organisms temperatures in warm blooded animals ~radiant energy is kinetic energy of photons (waves of light) [can be converted to thermal energy when light is absorbed by molecules and converted into motion] ~mechanical energy results from conversion of stored chemical energy [changes in lengths of cytoskeletal filaments generates forces that push or pull on membranes or organelles] ~electric energy is energy of moving electrons or other charged particles ~chemical potential energy- energy stored in bonds connecting atoms in molecules [high potential energy in covalent bonds of glucose can be release by controlled enzymatic combustion in cells] ~concentration gradient- when concentration of substance on one side of barrier is different from that on the other side [all cells form concentration gradients b/w interior and exterior fluids by selectively exchanging nutrients/ions] ~electric potential- the energy of charge separation

specific binding of ligands underlies the functions of most proteins

~ligand- molecule to which protein binds [ligand binding causes a change in shape of a protein] ~properties that characterize how protein binds to ligand- (1) specificity [ability of protein to bind one molecule or small group of molecules in preference to other molecules] (2) affinity [tightness or strength of binding expressed as dissociation constant Kd] ~stronger the interaction b/w protein and ligand the lower the value of Kd [both the specificity and affinity of protein for a ligand depend on structure of ligand binding site] ~molecular complementarity- for high affinity and highly specific interactions to take place the shape and chemical properties of binding site must be complementary to that of ligand molecule [molecular complementarity allows molecules to form multiple noncovalent interactions at close range and thus stick together] ~antibodies have characteristic of binding specifically to part of antigen called epitope [antibodies act as specific sensors for antigens forming antibody-antigen complexes that initiate cascade of protective reactions in cells of immune system] ~antibodies are Y shaped molecules (each arm contains a single light chain linked to a heavy chain by a disulfide bond) [end of each arm has 6 variable loops called complementarity-determining regions (CDRs) which form antigen binding sites] ~sequences of loops are highly variable generating unique complementary ligand binding sites that make them specific for different epitopes ~specificity of antibodies is so precise they can distinguish b/w proteins w/ identical sequences and only diff post translational modifications

liquid chromatography resolves proteins by mass, charge or binding affinity

~liquid chromatography- molecules dissolved in solution interact differently w/ solid surface [molecules that interact frequently w/ surface will spend more time bound to surface and thus flow past surface more slowly] [sample is placed on column of beads and flows down column by hydrostatic forces- nature of beads determines whether separation depends on mass, charge of binding affinity] ~gel filtration chromatography- the smaller the proteins mass the more time its trapped on beads and the greater the elution volume [by use of proteins of known mass the elution volume can be used to estimate mass of protein] ~ion exchange chromatography- when proteins flow through positively charged beads only proteins w/ negative charge adhere to beads and neutral and positive proteins flow through and vice versa ~affinity chromatography- ligands that bind to protein of interest are covalently attached to beads

NAD⁺ and FAD couple many biological oxidation and reduction reactions

~loss of electrons from atom/molecule is oxidation and gain of electron is reduction ~many biological oxidation and reduction reactions involve removal or addition of H atom (proton) rather than transfer of isolated electrons [this is b/c protons are soluble in aqueous solution but electrons arent and must be transferred directly from one molecule to another w/o water dissolved intermediate] ~in these types of reactions electrons are transferred to small electron carrying molecules called coenzymes [common coenzymes are NAD⁺ which is reduced to NADH and FAD which is reduced to FADH₂] [reduced forms of coenzymes can transfer protons and electrons to other molecules] ~readiness where molecule gains electron is reduction potential (E) and tendency to lose electrons is oxidation potential (which has the same magnitude but opposite sign as reduction potential for reverse reaction) ~in redox reaction electrons move spontaneously toward atom or molecules having more positive reduction potentials [molecule w/ more negative reduction potential can transfer electrons spontaneously to (reduce) a molecule w/ more positive reduction potential]

decoding of mRNA by tRNAs

~mRNA- carries genetic info transcribed from DNA in linear form [mRNA is read in sets of 3 nucleotide sequences (codons) each which specifies a particular amino acid] ~tRNA- each type of amino acid has its own subset of tRNAs which bind amino acid and carry it to growing end of polypeptide chain when next codon in mRNA calls for it [tRNA contains anticodon that can base pair w/ its complementary codon in mRNA] ~rRNA- associates w/ set of proteins to form ribosomes [ribosomes are comprised of large and small subunit each of which contains its own rRNA molecule] ~polypeptide chains resulting from translation undergo post translational folding and often other changes (chemical modifications) that are required for production of functional protein

all eukaryotic cells utilize a similar cycle to regulate their division

~mitosis- chromosomes are duplicated during S phase and the replicated chromosomes separate during M phase with each daughter cell getting a copy of each chromosome during cell division [S and M phases are separated by 2 gap phases (G1 and G2 phases) during which mRNAs and proteins are made and cell increases in size]

monosaccharides covalently assemble into linear and branched polysaccharides

~monosaccharides are carbohydrates which are covalently bonded combos of carbon and water in a 1:1 ratio [(CH₂O)n where n=3,4,5,6 or 7] [hexoses (n=6) and pentoses (n=5) are most common monosaccharides] ~all monosaccharides contain hydroxyl group and either aldehyde or keto group ~D-glucose is the principal source of energy for most cells and can exist in 3 forms- linear structure and 2 diff hemiacetal ring structures [to make the hemiacetal ring of D-glucopyranose the aldehyde group on carbon 1 combines w/ hydroxyl group on carbon 5] [α D-glucopyranose the hydroxyl group points downward from the ring and in β it points upward] ~in aqueous solution α and β readily interconvert spontanteously (at equilibrium there is 1/3 α and 2/3 β with very little of the open chain form)

multiple polypeptides assemble into quaternary structures and supramolecular complexes

~multimeric proteins consist of 2 or more polypeptide chains (subunits) ~quaternary structure- describes number and relative positions of the subunits in multimeric proteins [can be composed of various identical (homomeric) or different (heteromeric) subunits] ~individual monomer subunits of multimeric protein cant function normally unless they are assembled into multimeric protein ~metabolic coupling- assembly into multimeric protein permits proteins that act sequentially in a pathway to increase their efficiency of operation owing to their juxtaposition in space ~supramolecular complexes- containing tens to hundreds of polypeptide chains and sometime other biopolymers such as nucleic acids [act as molecular machines carrying out the most complex cellular processes by integrating multiple proteins each w/ distinct functions into one large assembly]

nonsense mutations cause premature termination of protein synthesis

~nonsense mutations- mutation that causes base pair change that converts normal codon into stop codon [effect of nonsense mutation can be suppressed by second mutation in tRNA gene] ~cells w/ original nonsense mutation and second mutation in anticodon of tRNA can insert tyrosine at position of mutant stop codon allowing protein synthesis to continue past nonsense mutation

five different nucleotides are used to build nucleic acids

~nucleotides all have common structure- phosphate group linked by phosphodiester bond to pentose (5 carbon sugar) that is linked to N and C containing ring structure referred to as the base ~in RNA the pentose sugar is ribose and in DNA the pentose sugar is deoxyribose ~adenine and guanine are purines which contain pair of fused rings while cytosine/thymine/uracil are pyrimidines which contain single ring ~1' C atom of sugar is attached to N at position 9 of purine and position 1 of pyrimidine ~acidic character of nucleotides is due to phosphate group which releases protons leave phosphate negatively charged ~nucleosides- combinations of a base and sugar w/o phosphate [nucleotides are nucleosides that have 1/2/3 phosphate groups esterified at 5' hydroxyl

chemical reactions in cells are at a steady state

~within cells many reactions are linked in pathways where product has alternative fates besides reforming reactant [can be used as reactant in another reaction or pumped out of the cell] ~steady state- in non equilibrium conditions the rate of formation of a substance can be equal to its rate of consumption [so concentration of substance remains constant over time] ~consequence of steady state is that it prevents accumulation of excess intermediates protecting cell from harmful effects of toxic intermediates at high concentrations

template DNA strand is transcribed into complementary RNA chain by RNA polymerase

~one DNA strand acts as template determining order in which rNTP monomers are polymerized to form complementary RNA chain [bases in template strand base pair w/ complementary incoming rNTPs which are joined in reaction catalyzed by RNA polymerase] [RNA molecules are always synthesized in 5' to 3' direction] ~energetics of polymerization favor addition of rNTPs to growing chain b/c high energy bond of phosphates of rNTPs is replaced by lower energy phosphodiester bond b/w nucleotides [pyrophosphatase catalyzes cleavage of PPi into 2 molecules of Pi which releases energy] ~site on DNA where RNA polymerase begins transcription is +1 [downstream denotes direction in which template DNA is transcribed] ~b/c RNA is synthesized 5' to 3' RNA polymerase moves down template strand in 3' to 5' direction [RNA is identical to nontemplate DNA strand w/ U instead of T]

organization of genes differ in prokaryotic and eukaryotic DNA

~operon- contiguous array of genes that code for proteins w/ similar functions [operates as unit from single promoter] ~b/c DNA is not sequestered in nucleus in prokaryotes ribosomes have immediate access to translation start sites in mRNA as they emerge from RNA polymerase [translation of mRNA begins even while the 3' end of mRNA is still being synthesized at active site of RNA polymerase] ~economic clustering of genes devoted to single metabolic function doesnt occur in eukaryotes [each gene is transcribed from its owen promotor producing one mRNA which generally is translated to yield single polypeptide] ~gene is first transcribed into long primary transcript that includes both exon and intron sequences [exons are then spliced together removing introns] [introns are common in multicellular eukaryotes but rare in prokaryotes and unicellular eukaryotes]

eukaryotic translation initiation usually occurs at first AUG closest to 5' end of mRNA

~overall initiation: small and large subunits assemble around mRNA that has activated initiator tRNA correctly positioned at start codon in P site [in eukaryotes this process is mediated by eIFs] [as each component joins complex it is guided by interactions with eIFs and several of these initiation factors bind GTP and hydrolysis of GTP to GDP functions as proofreading switch] ~(1) 43S preinitiation complex is formed when 40S subunit w/ eIFs 1, 1A and 3 associates w/ eIF5 and Met-tRNA [eIF2 can only bind Met-tRNA when it is associated w/ GTP] (2) mRNA is activated when eIF4 complex binds to 5' cap (3) eIF4A RNA helicase unwinds RNA secondary structure as 40S scans in 5' to 3' direction until it recognizes initiation codon (4) recognition causes eIF5 to stimulate hydrolysis of GTP [switches conformation of scanning complex to initiation complex w/ anticodon of Met-tRNA base paired to AUG in P site] (5) 60S subunit joins 40S [formation of 80S w/ Met-tRNA in P site] ~in bacteria- Shine Dalgarno sequence precedes AUG by 4-7 nucleotides [ribosome recognizes Shine Dalgarno and puts it in proper position for initiation]

mass spectrometry can determine mass and sequence of proteins

~permits determination of ratio of mass (m) of charged molecule (molecular ion) to its charge (z) ~key features- (1) ion source [where charge is transferred to peptide or protein molecules] (2) mass analyzer [physically separates ions on basis of m/z ratios] (3) detector [provides measure of relative abundances of each of ions in sample] ~abundances of ions determined by mass spectrometry in any given sample or relative values [so if you want to compare different samples you have to have an internal standard in the samples whose amounts dont differ b/w 2 samples]

phospholipids associate noncovalently to form basic bilayer structure of biomembranes

~phospholipids consist of 2 long chain nonpolar fatty acid groups linked by an ester bond to small highly polar groups (including a phosphate and short organic molecule such as glycerol) ~fatty acids consist of hydrocarbon (acyl) chain attached to a carboxyl group (-COOH) [differ in length but have even number of carbon atoms] ~fatty acids are designated by the abbreviation Cx:y where x is number of carbons in chain and y is number of double bonds ~fatty acids are covalently attached to another molecule by dehydration reaction called esterification [in which OH from carboxyl group of fatty acid and H from hydroxyl group on other molecule are lost] [in the combined molecule the part from the fatty acid is called the acyl group] ~phosphoglycerides contain 2 acyl groups attached to 2 of the hydroxyl groups of glycerol ~fatty acyl groups can also be covalently linked into other fatty molecules including triglycerides (which contain 3 acyl groups esterfied to glycerol) and covalently attached to cholesterol to form cholesteryl esters ~consequence of unsaturated fatty acid (double bond) is that they can be cis or trans [cis double bond makes kink in otherwise flexible straight acyl chain] ~unsaturated fatty acids contain only cis bonds [saturated fatty acids w/o kink can pack together tightly so have higher melting points than unsaturated fatty acids] [saturated fatty acids are solid at room temp and unsaturated are liquid]

phosphorylation and dephosphorylation covalently regulate protein activity

~phosphorylation- reversible addition of phosphate groups to hydroxyl groups on side chains of residues [catalyzed by enzymes called protein kinases while dephosphorylation is catalyzed by phosphatases] ~counteracting activities of kinases and phosphatases provide cells w/ switch that can turn on/off function of various proteins ~phosphorylation changes proteins charge and can lead to conformational change that significantly alters ligand binding or other features of protein causing increase/decrease in activity ~target of kinase (and phosphatase) is yet another kinase/phosphatase creating a cascade effect [kinase cascades permit amplification of signal and many levels of fine tuning control]

different types of RNA exhibit various conformations related to their functions

~presence of T in DNA is important to long term stability of DNA b/c of its function in DNA repair ~most cellular RNAs are single stranded and exhibit variety of conformations [differences in conformations allow RNA to carry out specific functions] ~hairpins are formed by pairing of bases within 5-10 nucleotides of each other and stem loops by pairing of bases that are separated by >10 to several hundred nucleotides ~some ribozymes can catalyze splicing (which internal RNA sequence is cut and removed and resulting chains are ligated) [some RNAs carry out self splicing w/ catalytic activity residing in sequence that is removed]

enzymes in common pathway are often physically associated with one another

~products from one reaction can move by diffusion to next enzyme in pathway [diffusion entails random movement and can be slow relatively inefficient process for moving molecule b/w enzymes] ~metabolic coupling- mechanism for bringing enzymes in common pathway into close proximity [polypeptides w/ diff catalytic activities cluster together as subunits on common scaffold that holds them together] [this arrangement allows products of one reaction to be channeled directly to next enzyme in pathway]

the proteasome is molecular machine used to degrade proteins

~proteasomes consist of 50 protein subunits and have cylindrical catalytic core called 20S proteasome [bound to this core is either 1 or 2 19S cap complexes that regulate activity of core] [when core and caps are combined they are both referred to as 26S complex] [19S cap has 16-18 protein subunits 6 of which can hydrolyze ATP to provide energy needed to unfold protein substrates and transfer them into inner chamber of proteasome core] ~proteosomes can degrade most proteins thoroughly b/c the 3 active sites in each subunit ring can cleave hydrophobic resides, acidic residues and basic residues ~substrates enter chamber via regulated aperture (allows entry of only unfolded protein) and is controlled by ATPases in 19S cap ~short peptide residues of proteasomal digestion exit the chamber and are further degraded by cytosolic peptidases being converted to individual amino acids

hierarchical structure of proteins

~protein chain folds into distinct 3D shape that is stabilized by noncovalent interactions b/w regions in the linear sequence of amino acids ~function is derived from 3D structure and 3D structure is determined by both proteins amino acid sequence and intramolecular noncovalent interactions ~primary structure- linear sequence of amino acids linked together by peptide bonds ~secondary structure- folding of polypeptide chain into local α helices or β sheets ~tertiary structure- secondary structural elements together w/ various loops and turns in a single polypeptide chain pack into larger independently stable structure which may include distinct domains ~quaternary structure- some proteins consist of more than one polypeptide associated together ~organisms function so efficiently b/c they are comprised of many diff types of macromolecular machines [size of these machines falls in 1-100 nm range] [research on these machines is called nanotechnology] ~advantages of assembly of proteins into multi protein complexes- localization, efficiency, specificity, regulation

proteins give cells structure and perform most cellular tasks

~protein chains range in length from 100 to 1000 amino acids ~during polymerization a linear chain folds into complex shape giving a distinctive 3D structure and function for each protein ~most proteins are enzymes (which catalyze chemical reactions involving small molecules or macromolecules) [catalyze steps in synthesis of proteins or other macromolecules like DNA and RNA] ~cytoskeletal proteins serve as structural components of a cell and power the movement of subcellular structures such as chromosomes or even whole cells by using the energy stored in chemical bonds of ATP ~can bind adjacent cells together to form parts of extracellular matrix, sensors that change shape as temp/ion concentrations/other properties of cell change, can import/export (when in cell membrane) variety of small molecules and ions, can be hormones or hormone receptors, bind to specific segments of DNA to turn genes off/on ~how can 20 amino acids form all diff proteins? if you assume each protein is 400 amino acids long then 20^400 is astronomical

alternatively folded proteins are implicated in diseases

~protein may fold into alternative 3D structure as result of mutations and misfolding not only leads to loss of normal function of protein but often marks it for degradation ~when degradation isnt complete or doesnt keep pace with misfolding the accumulation of misfolded fragments contributes to certain degenerative diseases characterized by presence of insoluble aggregates of twisted together proteins (plaques) in various organs

purifying, detecting and characterizing proteins

~protein must be purified before its structure/mechanism of action can be studied in detail [b/c proteins vary in size/shape/charge/water solubility no one single method can isolate all proteins] ~any molecule can be resolved (separated) from other molecules on basis of their differences in one or more physical or chemical characteristics ~most widely used characteristics for separating proteins are size (length or mass), net electrical charge and binding affinity for specific ligands

proteomics

~proteomics is study of amounts, modifications, interactions, localization, and functions of all or subsets of proteins at whole organism level ~questions asked in proteomics: (1) what fraction of whole proteome is expressed in given sample (2) of proteins present what are their relative abundances (3) what are amounts of different splice forms and chemically modified forms (4) which proteins are present in multiprotein complexes and how do they interact (5) when the state of cell changes how do proteins change (6) can protein changes be used for diagnostic purposes (7) can changes in proteome help define targets for drugs

primary structure of protein is its linear arrangement of amino acids

~repeated amide N, α carbon, carbonyl C and oxygen atoms of each amino acid residue form the backbone of protein molecule from which various side chains project ~backbone exhibits directionality from N to C orientation [one end of protein has free unlinked amino group (N) and the other end has free carboxyl group (C)] [written N terminal on left and C terminal on right]

ribosomes are protein synthesizing machines

~ribosome is composed of 3 (bacteria) or 4 (eukaryotes) different rRNA molecules and as many as 83 proteins organized into small and large subunits [assembled ribosome is 70S in bacteria and 80S in eukaryotes] ~sites where tRNAs are bound by ribosomes are known as A, P and E sites [tRNAs move b/w these sites as protein synthesis takes place]

enzymes are highly efficient and specific catalysts

~ribozymes- catalytic RNA macromolecules ~although most enzymes are located within cells some are secreted and function at extracellular sites such as blood ~enzymes increase rate of reaction but dont affect extent of reaction (which is determined by change in ∆G b/w reactants and products) ~enzymes increase reaction rate by lowering energy of transition state and therefore activation energy required to reach it

protein conformation is determined by sophisticated physical methods

~x ray cyrstallography- beams of x rays are passed through protein crystal [electrons in atoms of crystal scatter the x rays which produces a diffraction pattern] ~cryoelectron microscopy- protein sample is rapidly frozen in liquid helium to preserve structure and then examined in frozen state in cryoelectron microscope [computer programs analyze images and reconstruct proteins structure in 3D] ~NMR spectroscopy- protein solution is placed in magnetic field and effects of radio frequencies on nuclear spin states of atoms are measured [determines distances b/w atoms]

serine proteases demonstrate how enzymes active site works

~serine protease mechanism points out features of enzymatic catalysis- (1) enzyme catalytic sites have evolved to stabilize the binding of transition state thus lowering activation energy and accelerating overall reaction (2) multiple side chains together w/ polypeptide backbone work together to chemically transform substrate into product often by multistep reactions (3) acid base catalysis mediated by one or more amino acid side chains is often used by enzymes [often only a particular ionization state of one or more amino acid side chains in catalytic site is compatible w/ catalysis and thus enzymes activity is pH dependent] ~pH sensitivity of enzymes activity can be due to changes in ionization of catalytic groups, groups that participate directly in substrate binding or groups that influence conformation of protein ~cofactor/prosthetic group- helper group is nonpolypeptide ion or molecule that is bound in active site and play essential role in reaction mechanism [some of these are chemically modified during reaction and need to be replaced/regenerated after each reaction] ~enzyme inhibitors- small molecules that can bind to active sites and disrupt catalytic reactions [competitive inhibitors- directly bind to enzymes binding site and directly compete w/ substrates ability to bind] [noncompetitive inhibitors- interfere by binding to some other site on enzyme and changing its conformation]

polysomes and rapid ribosome recycling increase efficiency of translation

~simultaneous translation of single mRNA by multiple ribosomes and rapid recycling of ribosomal subunits increase overall rate at which cells synthesize proteins ~polyribosomes/polysomes- mRNA attached to multiple ribosomes bearing growing polypeptide chain [multiple copies of poly(A) binding protein interact w/ both mRNA poly(A) tail and eIF4 (and since eIF4 also binds to 5' cap the 2 ends of mRNA are bridged together by proteins forming circular mRNA)] [the circular pathway enhances ribosome recycling and thus increases efficiency of protein synthesis]

nucleic acid strand is linear polymer with end to end directionality

~single nucleic acid strand has backbone composed of repeating pentose-phosphate units from which purine and pyrimidine bases extend as side groups [nucleic acid strand has end to end chemical orientation- 5' end has phosphate group on terminal sugar and 3' end has hydroxyl group] [so this means 5' to 3' directionality of nuclei acid strand] ~phosphodiester bond actually consists of 2 phosphoester bonds one on 5' side of phosphate and other on 3' side

chemical foundations

~small molecules and ions make up 7% of weight of living matter combining into larger macromolecular assemblies that make up cells machinery/architecture ~small molecules include amino acids, nucleotides, lipids and sugars ~amphipathic molecules- contain both hydrophobic and hydrophilic regions ~molecular complementarity lies at heart of all biomolecular interactions [2 proteins w/ complementary shapes come together to form tightly bound complex] ~source of energy for chemical reactions is hydrolysis of ATP [this energy is released when high energy phosphoan-hydride bond linking β and γ phosphates in ATP molecule is broken by addition of water molecule forming ADP and Pi] ~rigid planarity imposed by double bonds has enormous significances for shapes and flexibility of biomolecules ~diff stereoisomers of molecule usually have completely diff biological activities b/c arrangement of atoms within their structures (and thus their ability to interact w/ other molecules differs)

radioisotopes are indispensable tools for detecting biological molecules

~specific activity- amount of radioactivity per unit of material measured in disintegrations per minute [shorter the half life the higher its specific activity] ~enzymes cant distinguish b/w radioisotope labeled substrates and non-labeled substrates ~labeled compounds can be detected by autoradiography or by a "counter" [which one is used depends on nature of experiment] ~autoradiographic studies of whole cells are crucial in determine intracellular sites where various macromolecules are synthesized and movement of these macromolecules within cells [in situ hybridization- autoradiographic assay for detecting specific isolated DNA or RNA sequences at specific tissue locations] ~pulse chase experiments- used to trace changes in intracellular location of proteins or modification of protein over time [often pulse chase experiments (where protein is detected by autoradiography after immunoprecipitation and SDS PAGE) are used to follow rate of synthesis, modification and degradation of proteins by adding radioactive amino acid precursors during pulse and then detecting amounts and characteristics of radioactive protein during chase]

secondary structures are core elements of protein architecture

~stable spatial arrangements of segments of polypeptide chain held together by H bonds b/w backbone amide and carbonyl groups and often involving repeating structural patterns ~single polypeptide may contain multiple types of secondary structure in various portions of chain depending on sequence ~parts of polypeptide that dont form α helix, β sheet or β turn but still have well defined stable shape have irregular structure ~random coil- highly flexible parts of polypeptide chain that have no fixed 3D structure ~α helix- backbone forms spiral structure where carbonyl O is H bonded to amide H of amino acid four residues farther along the chain in direction of C terminus [within helix all backbone amino and carboxyl groups are H bonded to one another except at very beginning and end of helix] [directionality of helix causes all H bond acceptors to have same orientation pointing downwards resulting in a structure where there is a complete turn of the spiral every 3.6 residues] ~β sheet- each β strand is a short (5-8 residue) polypeptide segment that laterally packs with other β strands to make β sheet [H bonds occur b/w backbone atoms in separate but adjacent β strands and are oriented perpendicularly to chains of backbone atoms] [side chains stick out above and below the plane of the sheet] [β strands can be oriented in the same (parallel) or alternating (anti-parallel) directions w/ respect to each other in regards to N-C directionality] ~β turns- composed of 4 residues and located on surface of protein and form sharp bends that reverse direction of polypeptide backbone (often toward proteins interior) [stabilized by one H bond b/w end residues] [help large proteins fold into highly compact structures]

structural motifs are regular combinations of secondary structures

~structural motif- particular combination of 2 or more secondary structures that form distinct 3D shape [often associated w/ a specific function] ~coiled coil- many proteins assemble into dimers or trimers by using a coiled coil motif where α helices from 2, 3 or 4 separate polypeptide chains coil about one another resulting in a coil of coils [individual helices bind b/c each helix has strip of alipathic side chains that interacts w/ similar strip in adjacent helix thus stabilizing assembly of multiple independent helices] ~b/c leucine frequently appears in fourth positions and hydrophobic side chains merge together like teeth of zipper so these structural motifs called leucine zippers ~other structural motifs contain α helices like calcium binding motif EF hand [contains 2 short helices connected by loop (helix turn helix motif) and is used for sensing calcium levels in cells] ~basic helix-loop-helix (bHLH)-structural motifs used for protein binding to DNA and regulation of gene activity ~zinc finger- contains three secondary structures (α helix and 2 β strands (anti-parallel)) that form fingerlike bundle held together by zinc ion ~presence of same structural motif in diff proteins w/ similar functions indicates these useful combos of secondary structures have been conserved in evolution

specificity of degradation (ubiquitin marks cytosolic proteins for degradation in proteasomes)

~targeting of specific proteins for proteasomal degradation is through substrate specificity of E3 ligase ~some E3 ligases are associated w/ chaperones that recognize misfolded proteins [chaperone-ubiquitination-proteasome system works in concert for protein quality control]

tertiary structure is overall folding of polypeptide chain

~tertiary structure is stabilized by hydrophobic interactions b/w nonpolar side chains [these stabilizing interactions are often weak so tertiary structure is not rigidly fixed but undergoes continual fluctuations- this variation in structure has imp consequences for function and regulation of proteins] ~disulfide bonds b/w side chains of cysteine residues covalently links regions of proteins thus restricting flexibility and increasing stability of tertiary structure ~oil drop model of protein conformation- amino acids w/ hydrophilic side chains tend to be on outer surfaces of proteins (to help make proteins soluble in aqueous solutions) and amino acids w/ hydrophobic side chains form water insoluble central core ~3 structural categories of proteins based on tertiary structure- (1) globular proteins [water soluble, compactly folded, spheroidal and mixture of secondary structures] (2) fibrous [large elongated stiff molecules, composed of polypeptide chain w/ tandem copies of short sequence that forms single repeating secondary structure, aggregate into large multiprotein fibers that dont dissolve in water] (3) integral membrane proteins [embedded within phospholipid bilayer of membranes]

eukaryotic precursor mRNAs are processed to form functional mRNAs

~transcription and translation occur concurrently in bacteria [in eukaryotic cells site of RNA synthesis (nucleus) is separated from site of translation (cytoplasm) but also pre-mRNAs must undergo RNA processing to yield functional mRNA] ~5' cap (7 methylguanylate) that is connected to terminal nucleotide of RNA by 5',5' triphosphate linkage [cap protects mRNA from enzymatic degradation, assists mRNA export to cytoplasm and is bound by protein factor that is required to begin translation] ~processing at 3' end- cleavage by endonuclease to yield free 3' hydroxyl group where poly(A) tail is added by poly(A) polymerase ~RNA splicing- internal cleavage of introns and stitching together of exons ~functional mRNAs produced by RNA processing retain noncoding regions referred to as 5' and 3' untranslated regions (UTRs) at each end

folded structure of tRNA promotes its decoding functions

~translation requires tRNAs and enzymes called aminoacyl-tRNA synthetases [tRNA molecule must become chemically linked to amino acid via high energy bond forming aminoacyl-tRNA] [the anticodon in tRNA then base pairs w/ codon in mRNA so activated amino acid is added to growing polypeptide chain] ~tRNA molecules are 70-80 nucleotides long, fold into stem loop arrangement, 4 stems are short double helices stabilized by base pairing, 3 of the 4 stems have loops containing 7 bases at their ends while remaining unlooped stem contains free 3' and 5' ends of chain, anticodon are at center of middle loop, 3' end of unlooped amino acid acceptor stem has sequence CCA [which is added after processing of tRNA]

ubiquitination and deubiquitination covalently regulate protein activity

~ubiquitination can involve attachment of single ubiquitin (monoubiquitination), addition of multiple single ubiquitin molecules to diff sites of target protein (multiubiquitination) or addition of polymeric chain of ubiquitins (polyubiquitination) ~multiple forms of ubiquitination result in generation of wide variety of recognition surfaces that participate in many protein-protein interactions w/ hundreds of proteins that contain many distinct ubiquitin binding domains (UBD) ~ubiquitination unrelated to protein degradation also can control diverse cell functions including repair of damaged DNA, metabolism, mRNA synthesis, defense against pathogens, cell division/cell cycle progression, cell signaling pathways, trafficking of proteins within cell, apoptosis

during chain elongation each incoming aminoacy-tRNA moves through 3 ribosomal sites

~various aminoacyl-tRNAs diffuse into A site but next step in translation only proceeds when tRNA anticodon base pairs w/ codon in coding region [when that occurs GTP is hydrolyzed and this promotes conformational change the leads to release of GDP and binding of aminoacyl-tRNA in A site] ~w/ initiating Met-tRNA at P site and second tRNA at A site the two react forming a peptide bond [this peptidyltransferase reaction is catalyzed by large subunit rRNA ~following peptide bond synthesis the ribosome translocates along mRNA distance equal to one codon [once translocation has occurred correctly the bound GTP is hydrolyzed which prevents ribosome from moving along RNA in wrong direction] [translocation moves Met-tRNA to E site and other tRNA to P site and A site is open and available to accept another tRNA complex]

non covalent interactions

~when solid salts dissolve in water the ions separate from one another and are stabilized by their interactions w/ water molecules ~in aqueous solutions simple ions (such as Na⁺, K⁺, Ca²⁺, Mg²⁺ and Cl⁻) are hydrated surrounded by a stable shell of water molecules held in place by ionic interactions b/w central ion and oppositely charged end of water dipole ~most ionic compounds dissolve in water b/c energy of hydration is greater than lattice energy that stabilizes crystal structure ~parts or all of aqueous hydration shell must be removed from ions when they interact directly w/ proteins ~increasing concentration of salts such as NaCl in a solution of biological molecules can weaken and even disrupt ionic interactions holding biomolecules together ~hydrogen bonds are both longer and weaker than covalent bonds b/w same atoms [strongest H bond is when donor atom, H and acceptor atom all lie in straight line]

single celled eukaryotes are used to study cell structure and function

~yeast is used because (1) they can be grown easily and cheaply in culture from single cell [such cell clones all have same genes and same biochemical properties] (2) can grow by mitosis both as haploids and diploids which makes isolating mutations in genes relatively straightforward (3) have sexual cycle that allows exchange of genes b/w cells [under starvation diploid cells undergo meiosis to form haploid daughter cells that are a and α cells] ~one way to see mutated proteins to isolate organisms w/ temp sensitive mutations- mutants cant grow at higher temps b/c mutated protein unfolds and is not functional [most readily done with haploid organisms since there is only copy of each gene so mutation will have immediate consequence] ~homologous- proteins from different organisms but w/ similar amino acid sequences [may have same or similar functions]