Biology Review 1/28/2011. Cell Structure. Biological Molecules (DNA & Proteins) Central Dogma. Prokaryotic cell DNA (no nucleus) Membrane

Transcription

1 Biology Review Cell Structure Biological Molecules (DNA & Proteins) Central Dogma Structure and Function of DNA Replication, Transcription, Translation Regulation of Gene Expression Eukaryotic cell Membrane Cytoplasm Prokaryotic cell DNA (no nucleus) Membrane Organelles Nucleus (contains DNA) 1 µm 1

2 Prokaryotic cell Fimbriae Cell wall Circular chromosome Capsule Sex pilus Internal organization Flagella ENDOPLASMIC RETICULUM (ER) Rough ER Smooth ER Flagellum Nuclear envelope Nucleolus Chromatin NUCLEUS Centrosome CYTOSKELETON: Microfilaments Intermediate filaments Microtubules Plasma membrane Ribosomes Microvilli Peroxisome Golgi apparatus Mitochondrion Lysosome Animal cell 2

3 Cell Component Structure Function The eukaryotic cell s genetic instructions are housed in the nucleus and carried out by the ribosomes Nucleus Surrounded by nuclear envelope (double membrane) perforated by nuclear pores. The nuclear envelope is continuous with the endoplasmic reticulum (ER). Houses chromosomes, made of chromatin (DNA, the genetic material, and proteins); contains nucleoli, where ribosomal subunits are made. Pores regulate entry and exit os materials. (ER) Ribosome Two subunits made of ribosomal RNA and proteins; can be free in cytosol or bound to ER Protein synthesis The endomembrane system regulates protein traffic and performs metabolic functions in the cell Cell Component Structure Function Endoplasmic reticulum Extensive network of membrane-bound tubules and (Nuclear sacs; membrane separates envelope) lumen from cytosol; continuous with the nuclear envelope. Smooth ER: synthesis of lipids, metabolism of carbohydrates, Ca 2+ storage, detoxification of drugs and poisons Rough ER: Aids in sythesis of secretory and other proteins from bound ribosomes; adds carbohydrates to glycoproteins; produces new membrane Golgi apparatus Stacks of flattened membranous sacs; has polarity (cis and trans faces) Modification of proteins, carbohydrates on proteins, and phospholipids; synthesis of many polysaccharides; sorting of Golgi products, which are then released in vesicles. Lysosome Vacuole Membranous sac of hydrolytic enzymes (in animal cells) Large membrane-bounded vesicle in plants Breakdown of ingested substances cell macromolecules, and damaged organelles for recycling Digestion, storage, waste disposal, water balance, cell growth, and protection 3

4 Cell Component Structure Function Mitochondria and chloroplasts change energy from one form to another Mitochondrion Bounded by double membrane; inner membrane has infoldings (cristae) Cellular respiration Chloroplast Typically two membranes around fluid stroma, which contains membranous thylakoids stacked into grana (in plants) Photosynthesis Peroxisome Specialized metabolic compartment bounded by a single membrane Contains enzymes that transfer hydrogen to water, producing hydrogen peroxide (H 2 O 2 ) as a by-product, which is converted to water by other enzymes in the peroxisome Overview: The Molecules of Life All living things are made up of four classes of large biological molecules: carbohydrates, lipids, proteins, and nucleic acids Within cells, small organic molecules are joined together to form larger molecules Macromolecules are large molecules composed of thousands of covalently connected atoms Molecular structure and function are inseparable 4

5 Macromolecules are polymers, built from monomers A polymer is a long molecule consisting of many similar building blocks These small building-block molecules are called monomers Three of the four classes of life s organic molecules are polymers: Carbohydrates Proteins Nucleic acids The Diversity of Polymers Each cell has thousands of different kinds of macromolecules Macromolecules vary among cells of an organism, vary more within a species, and vary even more between species An immense variety of polymers can be built from a small set of monomers 5

6 Proteins have many structures, resulting in a wide range of functions Proteins account for more than 50% of the dry mass of most cells Protein functions include structural support, storage, transport, cellular communications, movement, and defense against foreign substances 6

7 Proteins Proteins perform many functions: Structural roles: i.e. keratin and collagen Muscle contraction: i.e. actin and myosin Endocrine function: i.e. hormones Transport molecules in the blood (hemoglobin carries O2) Channel proteins. Immune function: i.e. antibodies Biological catalysts: i.e. Enzymes Enzymes are a type of protein that acts as a catalyst to speed up chemical reactions Enzymes can perform their functions repeatedly, functioning as workhorses that carry out the processes of life Etc. Proteins are made up of amino acids Polypeptides Polypeptides are polymers built from the same set of 20 amino acids A protein consists of one or more polypeptides 7

8 Amino Acid Monomers Amino acids are organic molecules with carboxyl and amino groups Amino acids differ in their properties due to differing side chains, called R groups α carbon Amino group Carboxyl group 8

9 Nonpolar Glycine (Gly or G) Alanine (Ala or A) Valine (Val or V) Leucine (Leu or L) Isoleucine (Ile or Ι) Methionine (Met or M) Serine (Ser or S) Threonine (Thr or T) Acidic Phenylalanine (Phe or F) Cysteine (Cys or C) Polar Electrically charged Trypotphan (Trp or W) Tyrosine (Tyr or Y) Asparagine (Asn or N) Basic Proline (Pro or P) Glutamine (Gln or Q) The twenty amino acids of proteins. The amino acids are grouped here according to the properties of the side chains (R groups) highlighted in white. The amino acids are shown in their prevailing ionic forms at ph 7.2 the ph within a cell. The three letter and more commonly used one letter abbreviations for the amino acids are in the parentheses. All of the amino acids used in proteins are the same enantiomer called the L form as shown here Aspartic acid (Asp or D) Glutamic acid (Glu or E) Lysine (Lys or K) Arginine (Arg or R) Histidine (His or H) Amino Acid Polymers Amino acids are linked by peptide bonds A polypeptide is a polymer of amino acids Polypeptides range in length from a few to more than a thousand monomers Each polypeptide has a unique linear sequence of amino acids 9

10 Peptide bond Fig (a) Peptide bond Side chains Backbone Making a polypeptide chain. A) Peptide bonds formed by dehydration reactions link the carboxyl group of one amino acid to the amino group of the next. B) The peptide bonds are formed one at a time with the amino acid at the amino end (N terminus). The polypeptide has a repetitive backbone (purple) to which the amino acid side chains are attached. (b) Amino end (N-terminus) Carboxyl end (C-terminus) Determining the Amino Acid Sequence of a Polypeptide The amino acid sequences of polypeptides were first determined by chemical methods Most of the steps involved in sequencing a polypeptide are now automated 10

11 Protein Structure and Function A functional protein consists of one or more polypeptides twisted, folded, and coiled into a unique shape The sequence of amino acids determines a protein s threedimensional structure A protein s structure determines its function Groove Groove a) A ribbon model shows how the single polypetide chain folds and coils to form the functional protein. (the yellow lines represent one type of chemical bond that stabilizes the protein s shape) b) A space-filling model of lysozyme Shows more clearly the globular shape seen in many proteins as well as the specific three dimensional structure unique to lysosyme. Structure of a protein, the enzyme lysozyme. Present in our sweat. Tears and saliva lysozyme is an enzyme that helps prevent infection by binding to and destroying specific molecules on the surface of many kinds of bacteria. The groove is the part of the protein that recognizes and binds to the target molecules on bacterial walls. 11

12 Levels of protein organization The structure of proteins has at least three levels of organization, and some can have four Primary structure- linear unique sequence of amino acids joined by peptide bonds. The primary structure of a protein is its unique sequence of amino acids Peptide bonds are polar and therefore the C=O of one amino acid can also H bond to the N-H of another amino acid, and a water molecule is formed Secondary structure- When the protein takes an orientation in space, a coiling of the chain gives rise to a helix whereas a folding of the chain leads to pleated sheets. H bonds between the peptide bonds hold the shape. Tertiary Structure- Tertiary structure is determined by interactions among various side chains (R groups) and consists of the final 3 D shape of the protein. This type of structure are maintained by : Covalent, ionic bonds between amino acid R groups Quaternary structure- If the protein is made up of more than one polypeptide chain i.e. hemoglobin It is critical that proteins have a certain structure (structure function relationship) It is crucial not to get denatured by changes in ph and temperature etc. Primary Structure Secondary Structure Tertiary Structure Quaternary Structure β pleated sheet + H 3N Amino end Examples of amino acid subunits α helix 12

13 Primary Structure Sequence of amino acids, it is like the order of letters in a long word Unique for each protein, encoded by DNA (determined by inherited genetic information) Two linked amino acids = dipeptide Three or more = polypeptide Backbone of polypeptide has N atoms: -N-C-C-N-C-C-N-C-C-N- 1 + H 3N 5 Amino end Primary Structure Amino acid subunits 20 + H 3 N Amino end Amino acid subunits Carboxyl end 13

14 Secondary Structure Secondary structure- When the protein takes an orientation in space, a coiling of the chain gives rise to a helix whereas a folding of the chain leads to pleated sheets. H bonds between the peptide bonds hold the shape The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone Typical secondary structures are a coil called an α helix and a folded structure called a β pleated sheet Secondary Structure β pleated sheet Examples of amino acid subunits α helix 14

15 Tertiary structure Tertiary structure is the final 3 D shape of the protein. Tertiary structure is determined by interactions between R groups, rather than interactions between backbone constituents These interactions between R groups include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions Strong covalent bonds called disulfide bridges may reinforce the protein s conformation Tertiary Structure Quaternary Structure 15

16 Hydrophobic interactions and van der Waals interactions Hydrogen bond Polypeptide backbone Disulfide bridge Ionic bond Polypeptide chain β Chains Iron Heme Collagen α Chains Hemoglobin 16

17 Quaternary Structure Quaternary structure- If the protein is made up of more than one polypeptide chain i.e. hemoglobin Hemoglobin is a globular protein consisting of four polypeptides: two alpha and two beta chains Collagen is a fibrous protein consisting of three polypeptides coiled like a rope What Determines Protein Structure? In addition to primary structure, physical and chemical conditions can affect structure Alterations in ph, salt concentration, temperature, or other environmental factors can cause a protein to unravel This loss of a protein s native structure is called denaturation A denatured protein is biologically inactive 17

18 Denaturation Normal protein Renaturation Denatured protein Denaturation and renaturation of a protein. High temperatures or various chemical treatments will denature a protein, causing it to lose its shape and hence its ability to function. If the denatured proteins remains dissolved it can often renature when the chemical and physical aspects of its environment are restored to normal Protein Folding in the Cell It is hard to predict a protein s structure from its primary structure Most proteins probably go through several states on their way to a stable structure Chaperonins are protein molecules that assist the proper folding of other proteins 18

19 Scientists use X-ray crystallography to determine a protein s structure Another method is nuclear magnetic resonance (NMR) spectroscopy, which does not require protein crystallization Bioinformatics uses computer programs to predict protein structure from amino acid sequences EXPERIMENT X-ray source X-ray beam Diffracted X-rays Crystal Digital detector X-ray diffraction pattern 19

20 Nucleic acids store and transmit hereditary information The amino acid sequence of a polypeptide is programmed by a unit of inheritance called a gene Genes are made of DNA, a nucleic acid Nucleic Acids There are two types of nucleic acids: DNA (deoxyribonucleic acid) stores genetic information in the cell and organism-it replicates and gets transmitted to other cells when they divide and also when an organism reproduces. DNA provides directions for its own replication. DNA also directs synthesis of messenger RNA (mrna) and, through mrna, controls protein synthesis (occurs in ribosomes) therefore DNA codes for the amino acids in proteins RNA (ribonucleic acid) can function as an intermediary molecule which conveys DNAs instructions regarding the aa sequence in proteins (among many other roles) Structure: Both are made up of nucleotides ( a molecular complex of phosphate a pentose sugar and a Nitrogenous base) There are two types of nucleic acids: Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA) 20

21 DNA Synthesis of mrna in the nucleus NUCLEUS Movement of mrna into cytoplasm via nuclear pore Synthesis of protein mrna CYTOPLASM mrna Ribosome DNA RNA protein. In a eukaryotic cell, DNA in the nucleus programs protein production in the cytoplasm by dictating synthesis of the messenger RNA mrna. The cell nucleus is actually much larger relative to the other elements in this figure Polypeptide Amino acids The Structure of Nucleic Acids Nucleic acids are polymers called polynucleotides Each polynucleotide is made of monomers called nucleotides Each nucleotide consists of a nitrogenous base, a pentose sugar, and a phosphate group The portion of a nucleotide without the phosphate group is called a nucleoside (nitrogenous base + sugar) 21

22 5 C 5 end Nitrogenous bases Pyrimidines 3 C Nucleoside Nitrogenous base Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA) Purines 5 C 3 C Phosphate group (b) Nucleotide Sugar (pentose) Adenine (A) Guanine (G) 3 end (a) Polynucleotide, or nucleic acid Sugars Components of nucleic acids. A) A polynucleotide has a sugar phosphate backbone with variable appendages the nitrogenous bases. B) A nucleotide monomer includes a nitrogenous base, a sugar and a phosphate group. Without the phosphate group the structure is called a nucleoside. C) A nucleoside includes a nitrogenous base (purine or pyrimidine) and a five carbon sugar deoxyribose or ribose Deoxyribose (in DNA) (c) Nucleoside components: sugars Ribose (in RNA) Nucleotide Monomers There are two families of nitrogenous bases: Pyrimidines (cytosine, thymine, and uracil) have a single six-membered ring Purines (adenine and guanine) have a six-membered ring fused to a five-membered ring In DNA, the sugar is deoxyribose; in RNA, the sugar is ribose 22

23 Nucleotide Polymers Nucleotide polymers are linked together to build a polynucleotide Adjacent nucleotides are joined by covalent bonds that form between the OH group on the carbon of one nucleotide and the phosphate on the carbon on the next These links create a backbone of sugar-phosphate units with nitrogenous bases as appendages. Covalent bonds in backbone The sequence of bases along a DNA or mrna polymer is unique for each gene The DNA Double Helix A DNA molecule has two polynucleotides spiraling around an imaginary axis, forming a double helix In the DNA double helix, the two backbones run in opposite directions from each other, an arrangement referred to as antiparallel One DNA molecule includes many genes The nitrogenous bases in DNA pair up and form hydrogen bonds: adenine (A) always with thymine (T), and guanine (G) always with cytosine (C) 23

24 RNA Usually single stranded Four types of nucleotides Unlike DNA, contains the base uracil in place of thymine There are several types of RNA among them are: mrna, rrna and trna which are key players in protein synthesis Structure of Nucleotides in DNA Each nucleotide consists of Deoxyribose (5-carbon sugar) Phosphate group A nitrogen-containing base Four bases Adenine, Guanine, Thymine, Cytosine 24

25 Sugar phosphate backbone end Nitrogenous bases The structure of a DNA strand. Each nucleotide consists of a nitrogenous base (T, A, C or G), the sugar deoxyribose (blue) and a phosphate group (yellow). The phosphate of one nucleotide is attached to the sugar of the next resulting in a backbone of alternating phosphates and sugars from which the bases project. The polynucleotide strand has directionality from the 5 end (with the phosphate group) to the 3 end (with the OH group). 5 and 3 refer to the numbers assigned to the carbons in the sugar ring. Phosphate Sugar (deoxyribose) end Thymine (T) Adenine (A) Cytosine (C) Guanine (G) DNA nucleotide end Hydrogen bond end 1 nm 3.4 nm 0.34 nm (a) Key features of DNA structure end (b) Partial chemical structure end (c) Space-filling model The double helix. A) The ribbons in this diagram represent the sugar phosphate backbones of the two DNA strands. The helix is right handed curving up to the right. The two strands are held together by hydrogen bonds (dotted lines) between the nitrogenous bases which are paired in the interior of the double helix. B) For clarity, the two strands of DNA are shown untwisted in this partial chemical structure. Strong covalent bonds link the units of each strand, while weaker hydrogen bonds hold one strand to the other. Notice that the strands are antiparallel, meaning that they are oriented in opposite directions. C) The tight stacking of the base pairs is clear in this computer model. Van der Waals attractions between the stacked pairs play a major role in holding the molecule together. 25

26 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Watson-Crick Model DNA consists of two nucleotide strands Strands run in opposite directions Strands are held together by hydrogen bonds between bases A binds with T and C with G Molecule is a double helix 26

27 Watson-Crick Model 2-nanometer diameter overall 0.34-nanometer distance between each pair of bases 3.4-nanometer length of each full twist of the double helix In all respects shown here, the Watson Crick model for DNA structure is consistent with the known biochemical and x-ray diffraction data. The pattern of base pairing (A only with T, and G only with C) is consistent with the known composition of DNA (A = T, and G = C). Base paring in DNA. The pairs of nitrogenous bases in DNA double helix are held together by hydrogen bonds shown here as pink dotted lines Adenine (A) Thymine (T) Guanine (G) Cytosine (C) 27

28 Many proteins work together in DNA replication and repair DNA is two nucleotide strands held together by hydrogen bonds Hydrogen bonds between two strands are easily broken The relationship between structure and function is manifest in the double helix Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material Each single strand then serves as template for new strand The Basic Principle: Base Pairing to a Template Strand Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules 28

29 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. b) The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. c) The complementary nucleotides line up and are connected to form the sugar-phosphate backbones of the new strands. Each daughter DNA molecule consists of one parental strand (dark blue) and one new strand (light blue). A model for DNA replication: the basic concept. In this simplified illustration a short segment of DNA has been untwisted into a structure that resembles a ladder. The rolls of the ladder are the sugar phosphate backbones of the two DNA strands. The rungs are the pairs of nitrogenous bases. Simple shape symbolizes the four kinds of bases. Dark blue represents DNA strands present in the parental molecule; light blue represents newly synthesized DNA. Watson and Crick s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or conserved from the parent molecule) and one newly made strand Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new) 29

30 Three alternative models of DNA replication. Each short segment of double helix symbolizes the DNA within a cell. Beginning with a parent cell we follow the DNA for two generations of cellstwo rounds of DNA replication. Newly made DNA is light blue. Parent cell a) Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. b) Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, complementary strand. c) Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA. First replication Second replication DNA Replication Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication bubble A eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied 30

31 Origin of replication Parental (template) strand Daughter (new) strand Doublestranded DNA molecule Two daughter DNA molecules Replication bubble Replication fork 0.5 µm (a) Origins of replication in E. coli In the circular chromosome of E coli and many other bacteria only one origin of replication is present. The parental strands separate at the origin, forming a replication bubble with two forks. Replication proceeds in both directions until the forks meet on the other side resulting in two daughter DNA molecules. The TEM shows a bacterial chromosome with a replication bubble. Origin of replication Parental (template) strand Daughter (new) strand 0.25 µm Bubble Replication fork Two daughter DNA molecules a) In each linear chromosomes of eukaryotes, DNA replication begins when replication bubbles form at many sites along the giant DNA molecule of each chromosome. The bubbles expand as replication proceeds in both directions. Eventually the bubbles fuse and synthesis of the daughter strands is complete. b) This micrograph, shows three replication bubbles along the DNA of a cultured Chinese hamster cell (TEM). Origins of replication in E. coli and eukaryotes. The red arrows indicate the movement of the replication forks and thus the overall directions of DNA replication within each bubble 31

32 At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating Helicases are enzymes that untwist the double helix at the replication forks Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template Topoisomerase corrects overwinding ahead of replication forks by breaking, swiveling, and rejoining DNA strands Single-strand binding proteins stabilize the unwound parental strands Primase synthesizes RNA primers using the parental DNA as a template Topoisomerase. Breaks swivels and rejoins the parental DNA ahead of the replication fork relieving the strain caused by unwinding RNA primer Helicase unwinds and separates the parental DNA strands Some of the proteins involved in the initiation of DNA replication. The same proteins function at both replication forks in a replication bubble. For simplicity only one fork is shown 32

33 DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the end The initial nucleotide strand is a short RNA primer An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template The primer is short (5 10 nucleotides long), and the end serves as the starting point for the new DNA strand Synthesizing a New DNA Strand Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Most DNA polymerases require a primer and a DNA template strand Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells 33

34 Primase synthesizes an RNA primer at the ends of the leading strand and the Okazaki fragments DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments DNA pol I removes primer from the ends of the leading strand and Okazaki fragments, replacing primer with DNA and adding to adjacent ends DNA ligase joins the end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments The DNA Replication Complex The proteins that participate in DNA replication form a large complex, a DNA replication machine The DNA replication machine is probably stationary during the replication process Recent studies support a model in which DNA polymerase molecules reel in parental DNA and extrude newly made daughter DNA molecules 34

35 New strand end Template strand end end end Sugar Phosphate Base end DNA polymerase end Pyrophosphate Nucleoside triphosphate end end Incorporation of a nucleotide into a DNA strand. DNA polymerase catalyzes the addition of a nucleoside triphosphate to the 3 end of a growing DNA strand with the release of two phosphates Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate datp supplies adenine to DNA and is similar to the ATP of energy metabolism The difference is in their sugars: datp has deoxyribose while ATP has ribose As each monomer of datp joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate 35

36 Antiparallel Elongation The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication DNA polymerases add nucleotides only to the free end of a growing strand; therefore, a new DNA strand can elongate only in the to direction Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase 36

37 Synthesis of the leading strand during DNA replication. This diagram focuses on the left replication fork shown in the overview box. DNA polymerase III (DNA pol III) shaped like a cupped hand is closely associated with a protein called the sliding clamp that encircles the newly synthesized double helix like a doughnut. The sliding clamp moves DNA pol III along the DNA template strand. Parental DNA Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication 1) After RNA primer is made DNA pol III starts to synthesize the leading strand. Origin of replication RNA primer Sliding clamp DNA poll III 2) The leading strand is elongated continuously in the 5 3 direction as the fork progresses Strand Assembly Why the discontinuous additions? Nucleotides can only be joined to an exposed OH group that is attached to the 3 carbon of a growing strand. This is why we say that DNA and RNA synthesis occurs in the 5 to 3 direction 37

38 Continuous and Discontinuous Assembly As Reiji Okazaki discovered, strand assembly is continuous on just one parent strand. This is because DNA synthesis occurs only in the 5 to 3 direction. On the other strand, assembly is discontinuous: short, separate stretches of nucleotides are added to the template, and then enzymes fill in the gaps between them. Continuous and Discontinuous Assembly Strands can only be assembled in the 5 to 3 direction 38

39 Overview Origin of replication Leading strand Lagging strand Lagging strand 2 1 Overall directions of replication Leading strand 1) Primase joins RNA nucleotides into a primer. Template strand RNA primer 2) DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment 1. Okazaki fragment 3) After reaching the next RNA primer to the right DNA pol III detaches. Synthesis of the lagging strand 4) After fragment 2 is primed, DNA pol III adds DNA nucleotides until it reaches fragment 1 and detaches. 5) DNA pol I replaces the RNA with DNA, adding to the end of fragment 2. 6) DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. Overall direction of replication 7) The lagging strand in this region is now complete. 39

40 Overview Origin of replication Leading strand Lagging strand 1) Helicase unwinds the parental double helix Parental DNA 2) Molecules of singlestrand binding protein stabilize the unwound template strands DNA pol III 5) DNA pol III is completing synthesis of the fourth fragment. When it reaches the RNA primer on the third fragment it will dissociate move to the replication fork and add DNA nucleotides to the 3 end of the fifth fragment primer 3) The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 4) Primase begins synthesis of the RNA Primer primer for the fifth Okazaki fragment DNA pol III Lagging strand 4 Leading strand Lagging strand Overall directions of replication 3 DNA pol I A summary of bacterial DNA replication. The detailed diagram shows one replication fork, but as indicated in the overview (upper right) replication usually occurs simultaneously at two forks one at either end of the replication bubble. Viewing each daughter strand in its entirety in the overview, you can see that half of it is made continuously as the leading strand while the other half (on the other side of the origin) is synthesized in fragments as the lagging strand. 2 7) DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment 6) DNA pol I removes the primer from the 5 end of the second fragment and replaces it with DNA nucleotides that it adds one by one to the 3 end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar phosphate backbone with a free 3 end. 1 40

41 Proofreading and Repairing DNA Mistakes can occur during replication DNA polymerase can read correct sequence from complementary strand and, together with DNA ligase, can repair mistakes in incorrect strandproofreading activity DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides In mismatch repair of DNA, repair enzymes correct errors in base pairing DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA A thymine dimer distorts the DNA molecule. Nucleotide excision repair of DNA damage. A team of enzymes detects and repairs damaged DNA. This figure shows DNA containing a thymine dimer, a type of damage often caused by ultraviolet radiation. A nuclease enzyme cuts out the damaged region of DNA and a DNA polymerase (in bacteria DNA pol I) replaces it with nucleotides complementary to the undamaged strand. DNA ligase completes the process by closing the remaining break in the sugar phosphate backbone. DNA polymerase DNA ligase A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease Repair synthesis by a DNA polymerase fills in the missing nucleotides. DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. 41

42 Replicating the Ends of DNA Molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes The usual replication machinery provides no way to complete the ends, so repeated rounds of replication produce shorter DNA molecules End of parental DNA strands Leading strand Lagging strand Shortening of the ends of linear DNA molecules. Here we follow the end of one strand of a DNA molecule through two rounds of replication. After the first round the new lagging strand is shorter than its template. After a second round both the leading and lagging strands have become shorter than the original parental DNA. Although not shown here the other ends of these DNA molecules also become shorter. RNA primer Lagging strand Primer removed but cannot be replaced with DNA because no end available for DNA polymerase Last fragment New leading strand New leading strand Previous fragment Removal of primers and replacement with DNA where a end is available Second round of replication Further rounds of replication Shorter and shorter daughter molecules 42

43 Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecule. Telomeres are involved in protecting chromosomes It has been proposed that the shortening of telomeres is connected to aging. Since telomeric sequences shorten each time the DNA replicates telomeres are also thought to be a molecular "clock" that regulates how many times an individual cell can divide. After a certain number of divisions the telomere shrinks to a certain level thereby causing the cell to stop dividing.the cell s metabolism slows down, it ages, and dies. Telomeres Telomeres exist at the ends of a chromosome They contain multiple copies of a G rich sequence In humans this sequence is TTAGGG, which can be repeated several thousand times 43

44 If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist Telomerase Shay et al. found that cellular aging can be bypassed or put on hold by the addition of the enzymatic component of telomerase Telomerase is a ribonucleoprotein enzyme complex. It consists of two components Protein- enzyme known as reverse transcriptase (RT) RNA- serves as the template The RNA functions as a template for the reverse transcriptase. The RT as the name suggests uses RNA as template to make DNA. So in this case the RT adds nucleotides to the chromosomal endstelomeres- thereby extending them. It stabilizes telomere length by adding hexameric (TTAGGG) repeats onto the telomeric ends of the chromosomes 44

45 Telomerase function Normal cells undergo a certain number of cell divisions and then seize to divide. Most normal cells do not have this enzyme and thus they lose telomeres with each division. If these cells are grown in tissue culture with the enzyme telomerase Their telomeres get extended They can continue to divide hundreds of generations past the time they normally would stop dividing. In humans, telomerase is active in germ cells, in the vast majority of cancer cells and, possibly, in some stem cells, epidermal skin cells and follicular hair cells. A chromosome consists of a DNA molecule packed together with proteins The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein In a bacterium, the DNA is supercoiled and found in a region of the cell called the nucleoid 45

46 Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells Histones are proteins that are responsible for the first level of DNA packing in chromatin Chromatin packing in a eukaryotic chromosome This series of diagrams and transmission electron micrographs depicts a current model for the progressive levels of DNA coiling and folding. The illustration zooms out from a single molecule of DNA to a metaphase chromosome, which is large enough to be seen with a light microscope. DNA double helix (2 nm in diameter) 1) DNA, the double helix Shown here is a ribbon model of DNA, with each ribbon representing one of the sugar-phosphate backbones. As you will recall from Figure 16.7, the phosphate groups along the backbone contribute a negative charge along the outside of each strand. The TEM shows a molecule of naked DNA; the double helix alone is 2 nm across. Histones Nucleosome (10 nm in diameter) Histone tail 2) Histones. Proteins called histones are responsible for the first level of DNA packing in Histones chromatin. Although each histone is small containing about 100 amino acids the total mass of histone in chromatin approximately equals the mass of DNA. More than a fifth of a histone's amino acids are positively charged (lysine or arginine) and bind tightly to the negatively charged DNA. Four types of histones are most common in chromatin: H2A, H2B, H3, and H4. The histones are very similar among eukaryotes; for example, all but two of the amino acids in cow H4 are identical to those in pea H4. The apparent conservation of histone genes during evolution probably reflects the pivotal role of histones in organizing DNA within cells. The four main types of histones are critical to the next level of DNA packing. (A fifth type of histone, called H1, is involved in a further stage of packing) H1 3) Nucleosomes, or beads on a string (10-nm fiber). In electron micrographs, unfolded chromatin is 10 nm in diameter (the 10-nm fiber). Such chromatin resembles beads on a string - Each "bead" is a nucleosome the basic unit of DNA packing; the "string" between beads is called linker DNA. A nucleosome consists of DNA wound twice around a protein core composed of two molecules each of the four main histone types. The amino end (N-terminus) of each histone (the histone tail) extends outward from the nucleosome. In the cell cycle, the histones leave the DNA only briefly during DNA replication. Generally they do the same during gene expression, another process that requires access to the DNA by the cell's molecular machinery. Chapter 18 will discuss some recent findings about the role of histone tails and nucleosomes in gene regulation 46

47 Chromatin packing in a eukaryotic chromosome Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber 4) 30-nm fiber. The next level of packing is due to interactions between the histone tails of one nucleosome and he linker DNA and nudeosomes on either side. A fifth histone, H1, is involved at this level. These interactions cause the extended 10-nm fiber to coil or fold, forming a chromatin fiber roughly 30 nm in thickness, the 30-nm fiber. Although the 30-nm fiber is quite prevalent in e i nterphase nucleus, the packing arrangement of nudeosomes in this form of chromatin is still a matter of some debate 5) Looped domains (300-nm fiber) The 30-nm fiber, in turn, forms loops called looped domains attached to a chromosome scaffold made of proteins, thus making up a 300-nm fiber. The scaffold is rich in one type of topoisomerase, and Hl molecules also appear to be present Replicated chromosome (1,400 nm) 6) Metaphase chromosome In a mitotic chromosome, the looped domains themselves coil and fold in a manner not yet fully understood, further compacting all the chromatin to produce the characteristic metaphase chromosome shown in the micrograph above. The width of one chromatid is 700 nm. Particular genes always end up located at the same places in metaphase chromosomes, indicating that the packing steps are highly specific and precise Chromatin is organized into fibers 10-nm fiber DNA winds around histones to form nucleosome beads Nucleosomes are strung together like beads on a string by linker DNA 30-nm fiber Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber 300-nm fiber The 30-nm fiber forms looped domains that attach to proteins Metaphase chromosome The looped domains coil further The width of a chromatid is 700 nm 47

48 Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis Loosely packed chromatin is called euchromatin During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Histones can undergo chemical modifications that result in changes in chromatin organization For example, phosphorylation of a specific amino acid on a histone tail affects chromosomal behavior during meiosis 48