MB Lec 5 PDF - Molecular Biology Lecture Notes

From gene to protein Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2024/2025 Housekeeping – book chapters Chapters from Essential Cell Biology by Bruce Alberts, 6th Edition that cover material taught in the lectures. Focus...

From gene to protein Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2024/2025 Housekeeping – book chapters Chapters from Essential Cell Biology by Bruce Alberts, 6th Edition that cover material taught in the lectures. Focus on what was discussed in the lectures. - available on eduportal Lecture 1: plant and animal cell structure - organelles, metabolism, maccromolecules Chapter 1: The fundamental units of life Chapter 2: Chemical components of the cell Chapter 13: How cells obtain energy from food Chapter 14: Energy generation in mitochondria and chloroplasts Chapter 17: Cytoskeleton (excluding muscular contraction) Lecture 2: cell membrane structure/function, transmembrane transport, organelle degradation Chapter 11: membrane structure Chapter 12: transport across cell membranes Lecture 3: cell signaling Chapter 16: cell signaling Lecture 4: cell cycle Chapter 18: the cell-division cycle Lecture 5: central dogma: transcription and translation Chapter 2: Chemical components of the cell (focusing on amino acids) Chapter 4: Protein structure and function Chapter 7: From DNA to Protein: how cells read the genome Lecture 6: genetic material Chapter 5: DNA and chromosomes  The metaphase chromosome Characteristic species-specific features of chromosomes: ❑ number in each cell ❑ shape and organization Morphological features of the metaphase chromosome: ❑ chromosome length ❑ centromere position ❑ arm length ❑ short arm (p) ❑ long arm (q) ❑ banding pattern  The metaphase chromosome Structural components of a metaphase chromosome: ❑ two sister chromatids ❑ a primary constriction (centromere) ❑ telomeres ❑ other structures ❑ secondary constriction(s) ❑ trabant / satellite  Centromere A centromere: ❑ contains centromeric DNA and histone proteins, ❑ is the site of attachment of sister chromatids, ❑ divides both chromatids into segments called arms, ❑ contains two kinetochores which play an important role in karyokinesis.  Kinetochore A kinetochore is a protein structure shaped like a layered plate located on both sides of the centromere in a chromosome The spindle fibers (microtubules) of the mitotic spindle attach to the kinetochore to allow the movement of chromosomes in metaphase and anaphase during cell division  Types of chromosomes There are four major types of chromosomes determined by the position of the centromere: 1. Metacentric The centromere is located exactly halfway between the chromosome ends The chromosome arms are equal in length 2. Submetacentric The centromere is located near the center of the chromosome During metaphase and anaphase, the chromosome assumes the shape of the letter "L"  Types of chromosomes 3. Acrocentric The centromere is located near the end of the chromosome The chromosome has a long (q) and short (p) arm 4. Telocentric The centromere is located at the end of the chromosome, which is why it has only one pair of long (q) arms Types of chromosomes  Telomeres A telomere: ❑ is a section of DNA located at the ends of a chromosome ❑ has a protective protein complex that forms two loops, the D-loop and T-loop, at its ends ❑ does not contain any genes and does not encode any proteins ❑ protects the chromosome from damage during cell division  Karyotype A karyotype is the complete set of chromosomes in a somatic cell of a given species Characteristic for all individuals: ❑ of the same species ❑ of the same sex ❑ in good health All are susceptible to the same chromosomal aberrations The karyotype distinguishes: ❑ autosomes: the same chromosomes between the sexes ❑ allosomes (heterosomes): the sex chromosomes  The human karyotype A karyotype is the visualization of chromosomes during mitotic cell division Chromosomes are spread out on the equatorial plate during metaphase Classic karyotype Spectral karyotype  Karyogram The karyogram is formed by chromosomes arranged in homologous pairs (i.e., one from the mother and the other from the father) The homologous pairs are arranged in order depending on the size of the chromosomes and the position of respective centromeres Strings (classical methods) or colors (spectral methods) are helpful in identifying homologues Karyogram  Cental dogma The description of the flow of information involving genetic material was coined the "central dogma of molecular biology" by Francis Crick in 1957 Expressing, "once the information has been transferred to the protein, it can no longer get out" Even with the addition of modern insights, Crick’s original dogma remains valid  Cental dogma Each process involved in the flow of genetic information has been designated a name The process of making an exact copy of DNA from an original DNA molecule is called, "DNA replication” The process of copying DNA to produce a single strand of mRNA with a sequence corresponding to that of a strand of DNA is termed, "transcription” Transcription refers to how information is transcribed from DNA to RNA using the language of nucleotides  Cental dogma The process of converting the nucleotide sequence of an mRNA molecule into the amino acid sequence of a protein is called, "translation” The term "translation" refers to how the information written in the language of nucleotides is translated into the language of amino acids The process of creating a single-stranded copy of DNA from a single-stranded RNA molecule is called, "reverse transcription" Cental dogma  Genetic code The genetic code is the set of rules that determine how genetic information encoding the order of amino acids that build proteins is recorded in DNA The genetic code is the set of rules for recording genetic information Number of nucleotides - 4 Number of amino acids - 20  Genetic code Characteristics of the genetic code: ❑ triplet nature ❑ non-ambiguity ❑ degeneracy ❑ non-overlapping ❑ commaless ❑ polarity ❑ universality  Genetic code Triplet nature: ❑ Three nucleotides - codon - codes for one amino acid ❑ a codon is the fundamental information unit in a gene ❑ there are 64 possible codons; ❑ 61 encode for amino acids ❑ Three signal the completion of protein synthesis (i.e., stop codons) Unambiguity: a given codon in DNA or RNA always corresponds to only one amino acid Degeneracy: different codons, usually differing only in the third nucleotide, can encode for the same amino acid  Genetic code Non-overlapping: codons do not overlap and are read sequentially Commaless: there are no "commas" nor punctuation (i.e., no physical or chemical indications or intermediaries separating neighboring codons)  Genetic code Polarity: the order of amino acids in a protein corresponds to the order of codons in DNA or RNA (read 5' to 3') Universality: codons encode the same amino acids in all organisms Genetic code Transcription and translation of the genetic code  Amino acids Amino acids are organic compounds containing two functional groups: ❑ amino – R-NH2 ❑ carboxyl – R-COOH In addition to the functional groups, each amino acid contains: ❑ a hydrogen atom ❑ an organic side chain (R group) The structure of an amino acid Amphoteric nature of amino acids All amino acids are amphoteric compounds; they can exhibit both: ❑ acidic properties due to the presence of the carboxyl group (-COOH), ❑ basic properties due to the amino group (-NH2). glycine  Types of amino acids Classification is due to the nature of the substituent: ❑ chain ❑ cyclic ❑ containing a hydroxyl group (-OH) ❑ containing a thiol group (-SH) ❑ containing an amino group (-NH2) alanine serine tyrosine cysteine asparagine  Types of amino acids Grouping by side chain structure: ❑ chain (aliphatic) ❑ straight chain ❑ branched chain ❑ ring (cyclic) ❑ aromatic ring ❑ hetrocyclic ring  Types of amino acids Grouping by the polarity of the side chain Polar - having a charge at neutral pH (7): ❑ positive - basic amino acids ❑ negative - acidic amino acids Polar - lacking a charge at neutral pH (7) Non-polar or hydrophobic: ❑ aliphatic side chain ❑ cyclic side chain Types of amino acids  Types of amino acids Grouping by the location of the amino group relative to the carbon it is bound to in the amino acid molecule The Greek letters α, β, γ, δ and ε indicate the position of the amino group at a given carbon atom α-amino acids - the amino group is bound to the first carbon that is bound to the carboxyl group β-amino acids - the amino group is bound to the second carbon from the carboxyl group γ-amino acids - the amino group is bound to the third carbon from the carboxyl group Types of amino acids  Types of amino acids Sub-classification of alpha-amino acids (except glycine) based on the spatial arrangement of the amino group relative to the chiral/asymmetric carbon: ❑ L-amino acids (left-handed) - the amino group is located on the left side of the asymmetric carbon ❑ D-amino acids (right-handed) - the amino group is located on the right side of the asymmetric carbon ❑ These AAs are mirror images of each other (enantiomers) like the left and right hands of a person Types of amino acids  Types of amino acids Grouping by origin: ❑ natural amino acids - found in living organisms ❑ synthetic amino acids - not found in nature (e.g., β-methylphenylalanine) Grouping based on participation in the structure of proteins in living organisms: ❑ protein amino acids ❑ non-protein amino acids  Protein amino acids Elements of the peptide and protein structure of all living organisms, from microorganisms to humans In addition, there is a pool of free protein amino acids, which have such important biological functions as: ❑ synthesis of lipids and their derivatives (serine, glycine) ❑ transmission of nerve signals (glutamic acid, aspartic acid) ❑ transmission of hormonal signals (tyrosine) These are amino acids .  Non-protein amino acids Occur in the free state or are products of protein amino acid metabolism They do not form protein structures, but have many important functions in the body These include β, γ and δ amino acids The most important non-protein AAs are: ❑ γ-aminobutyric acid (GABA): the main inhibitory neurotransmitter in the nervous system ❑ ornithine and citrulline: intermediate metabolites of the urea cycle  Types of amino acids Grouping by site of origin: Essential (exogenous): ❑ cannot be synthesized in the body of humans and higher animals ❑ must be supplied through food with the appropriate protein composition The need for essential amino acids depends on the degree of physiological development of the body  Types of amino acids Non-essential amino acids (endogenous) are amino acids that are synthesized in the human and animal bodies Relatively exogenous amino acids can be synthesized in the human and animal bodies provided that there is an adequate amount of their exogenous precursors in the diet Such amino acids include tyrosine (formed from phenylalanine) and cysteine (formed from methionine)  The role of amino acids -amino acids are the basic units of all bacterial, plant and animal proteins Ones included in proteins (protein amino acids) always have the L configuration Only by bacteria and plants have the ability to synthesize all protein amino acids  Peptides Peptides are compounds formed by linking two or more amino acids by means of a peptide bond A peptide bond is formed by the condensation reaction between: ❑ an amino group of one molecule ❑ a carboxyl group of the other molecule The by-product is a water molecule The formation and breakdown (hydrolysis) of peptide bonds are enzymatically controlled (e.g., peptidyltransferase and peptidase) The peptide bond  The peptide bond Within the peptide bond, the carbon-nitrogen bond: ❑ is partially within the nature of a double bond ❑ is shorter than the typical single C - N bond distance The length of the bond: ❑ C ─ N is 0.145 nm ❑ C ═ N is 0.127 nm ❑ C ─ N of the peptide bond is 0.132 nm  The peptide bond The C ─ N peptide bond is flat and rigid (four atoms lie within the same plane) which strongly inhibits rotation around this bond (partial double bond nature)  Peptides By convention, peptide nomenclature: ❑ begins with the amino group (-NH2) of the first amino acid ❑ then lists the subsequent amino acid residues ❑ ends with the carboxyl group (COOH) of the final amino acid The amino acid sequence is directional and is written using three-letter abbreviations Peptides  Types of peptides Oligopeptides contain up to 10 amino acid residues: ❑ dipeptides (two amino acids) ❑ tripeptides (three amino acids) ❑ tetrapeptides (four amino acids) ❑ decapeptides (10 amino acids) Polypeptides are peptides containing 11 to 100 amino acids residues Proteins (macropeptides) are peptides containing more than 100 residues of amino acids in the peptide chain Peptides of biological significance Glutathione is present in cells in both oxidized and reduced forms with a role in scavenging oxygen free radicals Methionine and leucine enkephalins are neurotransmitters that, along with endorphins (α, β, γ), are among the endogenous opiods with analgesic effects Oxytocin stimulates uterine smooth muscle contractions Vasopressin stimulates water and sodium resorption in the renal tubules and raises blood pressure Peptides of biological significance Insulin lowers blood glucose levels by transporting glucose into cells, and its deficiency is the cause of type I diabetes Glucagon is an antagonist of insulin; it stimulates an increase in blood glucose (via glycogenolysis) Parathyroid hormone (PTH) is responsible for calcium and phosphate regulation Bradykinin is an inflammatory molecule and dilates blood vessels and lowers blood pressure  Proteins Proteins are compounds made up of one or more polypeptide chains Proteins are macromolecules with the most diverse structures and functions Each protein is comprised of a sequence containing some or all of the 20 basic amino acids Modification of proline, lysine and cysteine increases the number of amino acids found in proteins to 23  Categorization of proteins Due to: ❑ origin (viral, bacterial, plant, animal) ❑ biological functions (enzymatic, structural, hormonal, transport, storage, toxins, etc.) ❑ shape and solubility in water: ❑ globular (spherical) and soluble ❑ neutral proteins (albumin, globulins) ❑ acidic proteins (prolamins, gluteins) ❑ basic proteins (histones, protamines) ❑ fibrous (in the form of fibers) and insoluble (hair and nail keratin, collagen, elastin)  Protein structure Proteins have a hierarchical structure and the structure are delineated into: ❑ primary ❑ secondary ❑ tertiary ❑ quaternary  Primary structure The primary structure of a protein is determined by the order of amino acids in the polypeptide chain Most proteins contain between 100 and 2000 amino acids The order of amino acids is strictly defined and characteristic of each protein  Secondary structures Secondary structures can be: ❑ regular ❑ α-helix (spiral shape) ❑ β-pleated sheet (corrugated sheet shape) ❑ irregular (random coils) ❑ beta-hairpin turns ❑ omega-loops (Ω loop)  Secondary structures α-helix: ❑ the pitch of the α-helix is 5.4 Å ❑ the diameter of the α-helix is 23 Å ❑ is stabilized by hydrogen bonds ❑ occurs between the NH and CO groups of neighboring amino acids In proteins all α-helices are right-handed -helix structure  An -helix example Ferritin, an iron storage protein has a secondary structure that is 75% in the α-helix form  Secondary structure β-pleated sheet: Consists of at least two almost completely stretched fragments of polypeptide chains (β-strands) stabilized by hydrogen bonds Strand structures of the β-sheet can be formed by 10 or more β-strands in different configurations The β-sheet structure can be: ❑ parallel ❑ anti-parallel β-sheet structure  Parallel β-sheet Hydrogen bonds between the NH and CO groups connect each amino acid of one strand to two different amino acids of a neighboring strand  Antiparallel β-sheet Hydrogen bonds between NH and CO groups connect each amino acid of one strand with an amino acid on the antiparallel strand (on the same plane) A β-sheet example Fatty acid-binding protein  Turns and loop structures Turns and loops are always found on the surface of proteins They take part in interactions between proteins and other molecules In turns, polypeptide chains stabilize hydrogen bonds between the NH and CO of the amino acid residues of i and i+3  Turns and loops structures The loops are rigid structures that are responsible for reversing the direction of the polypeptide chain  Tertiary structure The tertiary structure is the three-dimensional spatial arrangement of amino acid residues which are distant from each other in the primary structure The spatial arrangement of proteins is stabilised by bonds: ❑ covalent - disulfide “bridges” ❑ non-covalent through intramolecular interactions ❑ hydrogen ❑ hydrophobic ❑ van der Waals ❑ ionic (salt bridges) Tertiary structure Tertiary structure  Tertiary structure Types of tertiary structures: ❑ globular proteins ❑ fibrous proteins  Tertiary structure Globular proteins assume an approximately spherical shape Can be made up of both α-helices and β-sheets  Tertiary structure Fibrous proteins can have elongated, filamentous or stick- like structures Protein folding patterns: ❑ Three α-helixes - collagen (component of skin, tendons, ligaments) ❑ Two α-helixes forming a ‘superhelix’ structure – α-keratin (component of hooves, nails and hair) Actin (myofibrils, microfilaments, actomyosin) occurs in two forms: ❑ globular – G-actin ❑ filamentous – F-actin Tertiary structure  Tertiary structure G-Actin F-Actin  Tertiary structure In proteins there are structures called domains Domain is a region of a protein's polypeptide chain that is self- stabilizing and that folds independently from the rest They have a specific function in the protein (enzyme proteins) Proteins can have the same domains, even though their overall tertiary structures are different Domain 1 Domain 2 2 Domains - examples  Quaternary structure The quaternary structure of a protein is the association of several protein subunits into a closely packed arrangement Each of the subunits has its own primary, secondary, and tertiary structure Many proteins contain functionally diverse subunits: ❑ regulatory subunits ❑ catalytic subunits  Quaternary structure The quaternary structure can be: ❑ simple (identical subunits) ❑ complex (many different subunits) In most cases the subunits are linked to each other by non- covalent bonds Quaternary structure Protein structure levels  Non-protein structures Cofactors are non-protein components of proteins that are needed by enzymes to catalyse specific chemical reactions Cofactors are subdivided into: ❑ coenzymes which are non-permanently bound to proteins via non-covalent bonds ❑ prosthetic groups which are bound permanently to proteins via covalent or coordinate covalent bonds Coenzymes and prosthetic groups can be: ❑ organic (e.g., sugars or lipids) ❑ inorganic (metal ions and small inorganic molecules)  Protein complexes Glycoproteins - contain covalently bound straight chain or branched oligosaccharides Phosphoproteins - contain threonine or serine residues esterified with phosphoric acid Lipoproteins - are proteins complexed with lipids Metalloproteins - contain ions of various metals that interact with the protein either ionically or by coordination Nucleoproteins - are proteins complexed with DNA or RNA  Enzymatic proteins Many organic coenzymes and prosthetic groups are vitamins or respective derivatives, which are essential for functions of the body A protein without a prosthetic group is an apoprotein (apo- enzyme) A protein with a coenzyme/prosthetic group is a holoprotein  Amphoteric properties Proteins have an electrical charge which is determined by: ❑ the number and availability of acid and base groups in the side chain ❑ the pH of the environment At the isoelectric point (pI), the number of acidic groups is equal to the number of basic groups; the protein does not move under the influence of an electric field  Amphoteric properties At a pH lower than the isoelectric point, a protein is cationic and moves towards a cathode At a pH higher than the isoelectric point, a protein is anionic and moves towards an anode The differential mobility of proteins under an electric field permits protein identification and isolation by electrophoresis  Solubility Most proteins dissolve well in water Protein solutions are colloids In water, proteins hydrate, that is, the dipoles of water bind to: ❑ the N and O atoms of the peptide bonds ❑ the polar groups of the side chains Each protein is surrounded by an aqueous coating which determines the degree of suspension of the protein in colloidal solution  Functions of proteins Enzymatic functions - regulating all steps of metabolism in the cell through the specific properties of each enzyme Transport functions: ❑ transport of small molecules and ions ❑ transport of hydrophobic compounds (e.g., steroid and thyroid hormones, free bilirubin) ❑ transport of metal cations (e.g., transferrin – iron; ceruloplasmin - copper) ❑ storage and exchange of compounds with the environment (e.g., haemoglobin - transport of O2 and CO2)  Functions of proteins Structural functions: ❑ structure of cell membranes, cytoskeleton and cellular compartments (collagen, elastin, actin, β-keratin) ❑ histones (key role in DNA packaging) Protective function: ❑ immune protection - protecting the body against pathogens (immunoglobulins against bacteria, viruses, etc.) ❑ participation in blood clotting (fibrinogen-fibrin) ❑ antifreeze proteins  Functions of proteins Receiving and transmitting chemical and physical signals Replicative, transcriptional and translational functions Motor functions - regulate processes related to movement (actin, myosin) Spare functions: ❑ ovalbumin provides a source of amino acids for the embryo ❑ ferritin stores iron in the liver ❑ some proteins can be used for energy  Literature Fundamentals of cell biology, B. Alberts, D. Bray, K. Hopkin Volume 2 Chapter 4 Structure and function of proteins Spatial structure and structure of proteins How proteins work