The Molecular Basis of Inheritance

Transcription

1 LEURE PRESENIONS For MPBELL BIOLOY, NINH EDIION Jane B. Reece, Lisa. Urry, Michael L. ain, Steven. Wasserman, Peter V. Minorsky, Robert B. Jackson hapter 16 he Molecular Basis of Inheritance Lectures by Erin Barley Kathleen Fitzpatrick Overview: Life s Operating Instructions In 1953, James Watson and Francis rick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DN DN, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in DN and reproduced in all cells of the body his DN program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits Figure 16.1 oncept 16.1: DN is the genetic material Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists he Search for the enetic Material: Scientific Inquiry When. H. Morgan s group showed that genes are located on chromosomes, the two components of chromosomes DN and protein became candidates for the genetic material he key factor in determining the genetic material was choosing appropriate experimental organisms he role of DN in heredity was first discovered by studying bacteria and the viruses that infect them Evidence hat DN an ransform Bacteria he discovery of the genetic role of DN began with research by Frederick riffith in 1928 riffith worked with two strains of a bacterium, one pathogenic and one harmless 1

2 When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DN Figure 16.2 EXPERIMEN Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells RESULS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells Evidence hat Viral DN an Program ells In 1944, Oswald very, Maclyn Mcarty, and olin MacLeod announced that the transforming substance was DN heir conclusion was based on experimental evidence that only DN worked in transforming harmless bacteria into pathogenic bacteria Many biologists remained skeptical, mainly because little was known about DN More evidence for DN as the genetic material came from studies of viruses that infect bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research Figure 16.3 Bacterial cell Phage head ail sheath ail fiber DN 100 nm In 1952, lfred Hershey and Martha hase performed experiments showing that DN is the genetic material of a phage known as 2 o determine this, they designed an experiment showing that only one of the two components of 2 (DN or protein) enters an E. coli cell during infection hey concluded that the injected DN of the phage provides the genetic information 2

3 Figure EXPERIMEN Phage Bacterial cell Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid dditional Evidence hat DN Is the enetic Material Batch 1: Radioactive sulfur ( 35 S) Batch 2: Radioactive phosphorus ( 32 P) DN Radioactive DN Phage DN entrifuge Pellet (bacterial cells and contents) It was known that DN is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 1950, Erwin hargaff reported that DN composition varies from one species to the next his evidence of diversity made DN a more credible candidate for the genetic material entrifuge Pellet Radioactivity (phage DN) in pellet Figure 16.UN04 wo findings became known as hargaff s rules he base composition of DN varies between species In any species the number of and bases are equal and the number of and bases are equal he basis for these rules was not understood until the discovery of the double helix Figure 16.5 Sugar phosphate backbone Nitrogenous bases end Building a Structural Model of DN: hymine () Scientific Inquiry Phosphate Sugar (deoxyribose) DN Nitrogenous base nucleotide end denine () ytosine () uanine () fter DN was accepted as the genetic material, the challenge was to determine how its structure accounts for its role in heredity Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the DN molecule using this technique 3

4 Figure 16.6 (a) Rosalind Franklin (b) Franklin s X-ray diffraction photograph of DN Franklin s X-ray crystallographic images of DN enabled Watson to deduce that DN was helical he X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases he pattern in the photo suggested that the DN molecule was made up of two s, forming a double helix Figure nm end 3.4 nm end 0.34 nm Hydrogen bond end end Watson and rick built models of a double helix to conform to the X-rays and chemistry of DN Franklin had concluded that there were two outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule s interior Watson built a model in which the backbones were antiparallel (their subunits run in opposite directions) (a) Key features of DN structure (b) Partial chemical structure (c) Space-filling model Figure 16.UN01 t first, Watson and rick thought the bases paired like with like ( with, and so on), but such pairings did not result in a uniform width Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray data Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data 4

5 Figure 16.8 Watson and rick reasoned that the pairing was more specific, dictated by the base structures hey determined that adenine () paired only with thymine (), and guanine () paired only with cytosine () he Watson-rick model explains hargaff s rules: in any organism the amount of =, and the amount of = Sugar Sugar denine () Sugar hymine () Sugar uanine () ytosine () oncept 16.2: Many proteins work together in DN replication and repair he relationship between structure and function is manifest in the double helix Watson and rick noted that the specific base pairing suggested a possible copying mechanism for genetic material he Basic Principle: Base Pairing to a emplate Strand Since the two s of DN are complementary, each acts as a template for building a new in replication In DN replication, the parent molecule unwinds, and two new daughter s are built based on base-pairing rules Figure (a) Parent molecule (b) Separation of s (c) Daughter DN molecules, each consisting of one parental and one new Watson and rick s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old (derived or conserved from the parent molecule) and one newly made ompeting models were the conservative model (the two parent s rejoin) and the dispersive model (each is a mix of old and new) 5

6 Figure (a) onservative model (b) Semiconservative model Parent cell First Second replication replication Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model hey labeled the nucleotides of the old s with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope (c) Dispersive model Figure EXPERIMEN 1 Bacteria cultured in medium with 15 N (heavy isotope) RESULS 3 DN sample 4 DN sample centrifuged centrifuged after first after second replication replication Bacteria transferred to medium with 14 N (lighter isotope) Less dense More dense ONLUSION Predictions: First replication Second replication 2 DN Replication: loser Look he copying of DN is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DN replication onservative model Semiconservative model Dispersive model etting Started Replication begins at particular sites called origins of replication, where the two DN s are separated, opening up a replication bubble 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 Figure 16.12a (a) Origin of replication in an E. coli cell Origin of replication Doubleed DN molecule wo daughter DN molecules Parental (template) Replication bubble Daughter (new) Replication fork 0.5 µm 6

7 Figure 16.12b (b) Origins of replication in a eukaryotic cell Double-ed Origin of replication DN molecule Parental (template) Daughter (new) t the end of each replication bubble is a replication fork, a Y-shaped region where new DN s are elongating Bubble Replication fork Helicases are enzymes that untwist the double helix at the replication forks Single- binding proteins bind to and stabilize single-ed DN wo daughter DN molecules 0.25 µm opoisomerase corrects overwinding ahead of replication forks by breaking, swiveling, and rejoining DN s Figure Primase opoisomerase RN primer DN polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the end he initial nucleotide is a short RN primer Helicase Single- binding proteins Synthesizing a New DN Strand n enzyme called primase can start an RN chain from scratch and adds RN nucleotides one at a time using the parental DN as a template he primer is short (5 10 nucleotides long), and the end serves as the starting point for the new DN Enzymes called DN polymerases catalyze the elongation of new DN at a replication fork Most DN polymerases require a primer and a DN template he rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells 7

8 Figure New emplate Each nucleotide that is added to a growing DN is a nucleoside triphosphate dp supplies adenine to DN and is similar to the P of energy metabolism he difference is in their sugars: dp has deoxyribose while P has ribose s each monomer of dp joins the DN, it loses two phosphate groups as a molecule of pyrophosphate Sugar Phosphate P P P OH OH Base Nucleoside triphosphate DN polymerase P P i OH Pyrophosphate 2 P i ntiparallel Elongation he antiparallel structure of the double helix affects replication DN polymerases add nucleotides only to the free end of a growing ; therefore, a new DN can elongate only in the to direction long one template of DN, the DN polymerase synthesizes a leading continuously, moving toward the replication fork Figure Leading Overview Origin of replication Lagging Lagging Parental DN Primer Overall directions of replication Leading Origin of replication RN primer Sliding clamp DN pol III o elongate the other new, called the lagging, DN polymerase must work in the direction away from the replication fork he lagging is synthesized as a series of segments called Okazaki fragments, which are joined together by DN ligase 8

9 Figure 16.16b-6 Figure emplate 1 RN primer for fragment 1 Leading Overview Origin of replication Lagging RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment Parental DN DN pol III Primer Primase Leading DN pol III 4 Lagging Overall directions of replication Lagging DN pol I Leading DN ligase Overall direction of replication he DN Replication omplex he proteins that participate in DN replication form a large complex, a DN replication machine he DN replication machine may be stationary during the replication process Recent studies support a model in which DN polymerase molecules reel in parental DN and extrude newly made daughter DN molecules Figure Parental DN onnecting protein DN pol III DN pol III Helicase Lagging Leading Lagging template Proofreading and Repairing DN DN polymerases proofread newly made DN, replacing any incorrect nucleotides In mismatch repair of DN, repair enzymes correct errors in base pairing DN can be damaged by exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DN Figure Nuclease DN polymerase DN ligase 9

10 Evolutionary Significance of ltered DN Nucleotides Error rate after proofreading repair is low but not zero Sequence changes may become permanent and can be passed on to the next generation hese changes (mutations) are the source of the genetic variation upon which natural selection operates Replicating the Ends of DN Molecules Limitations of DN polymerase create problems for the linear DN of eukaryotic chromosomes he usual replication machinery provides no way to complete the ends, so repeated rounds of replication produce shorter DN molecules with uneven ends his is not a problem for prokaryotes, most of which have circular chromosomes Figure Ends of parental DN s Leading Lagging Lagging Parental Last fragment RN primer New leading New lagging Next-to-last fragment Removal of primers and replacement with DN where a end is available Second round of replication Further rounds of replication Eukaryotic chromosomal DN molecules have special nucleotide sequences at their ends called telomeres elomeres do not prevent the shortening of DN molecules, but they do postpone the erosion of genes near the ends of DN molecules It has been proposed that the shortening of telomeres is connected to aging Shorter and shorter daughter molecules Figure If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce n enzyme called telomerase catalyzes the lengthening of telomeres in germ cells 1 µm 10

11 he shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions here is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist oncept 16.3 chromosome consists of a DN molecule packed together with proteins he bacterial chromosome is a double-ed, circular DN molecule associated with a small amount of protein Eukaryotic chromosomes have linear DN molecules associated with a large amount of protein In a bacterium, the DN is supercoiled and found in a region of the cell called the nucleoid Figure 16.22a hromatin, a complex of DN and protein, is found in the nucleus of eukaryotic cells hromosomes fit into the nucleus through an elaborate, multilevel system of packing DN double helix (2 nm in diameter) Nucleosome (10 nm in diameter) DN, the double helix Histones Histones Histone tail H1 Nucleosomes, or beads on a string (10-nm fiber) Figure 16.22b 30-nm fiber 30-nm fiber Loops Scaffold 300-nm fiber Looped domains (300-nm fiber) Replicated chromosome (1,400 nm) hromatid (700 nm) Metaphase chromosome hromatin undergoes changes in packing during the cell cycle t interphase, some chromatin is organized into a 10-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping hough interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus 11

12 Figure 16.UN03 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 DN pol III synthesizes leading continuously Parental DN Helicase Primase synthesizes a short RN primer DN pol III starts DN synthesis at end of primer, continues in direction Lagging synthesized in short Okazaki fragments, later joined by DN ligase DN pol I replaces the RN primer with DN nucleotides Origin of replication Figure 16.UN06 12