Becker's World of the Cell Tenth Edition Chapter 16 PDF

Becker’s World of the Cell Tenth Edition Chapter 16 The Structural Basis of Cellular Information: DNA, Chromosomes, and the Nucleus Lectures by Anna Hegsted, Simon Fraser University Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Structural Basis of Cellular Information: DNA, Ch...

Becker’s World of the Cell Tenth Edition Chapter 16 The Structural Basis of Cellular Information: DNA, Chromosomes, and the Nucleus Lectures by Anna Hegsted, Simon Fraser University Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Structural Basis of Cellular Information: DNA, Chromosomes, and the Nucleus • Cells possess a set of “instructions” that specify their structure, dictate their functions, and regulate their activities. • These instructions can be passed on faithfully to daughter cells. • Hereditary information is transmitted in the form of distinct units called genes. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Genes Consist of DNA • Genes consist of DNA that codes for functional products that are usually protein chains. • The information in a cell’s DNA molecules undergoes replication to generate two copies for distribution into each daughter cell. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Transcription and Translation • Instructions stored in DNA are transmitted in a two-stage process called transcription and translation. • Transcription: RNA is synthesized in an enzymatic reaction that copies information from DNA. • Translation: the base sequence of RNA is used to direct the synthesis of a polypeptide. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Flow of Information in Cells (1 of 2) Figure 16.1 The Flow of Information in Cells. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Flow of Information in Cells (2 of 2) Figure 16.1 The Flow of Information in Cells. Copyright © 2022 Pearson Education, Inc. All Rights Reserved 16.1 Chemical Nature of the Genetic Material • In 1869, Johann Friedrich Miescher reported the discovery of the substance now known as DNA. • A few years later, Walther Flemming first observed chromosomes while studying dividing cells under the microscope. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Discovery of DNA Led to Conflicting Proposals Concerning the Chemical Nature of Genes • Miescher extracted a material from white blood cells that he called “nuclein,” now called DNA. • In the early 1880s, a botanist named Eduard Zacharias found that removing DNA from cells abolished the staining of chromosomes. • He and others began to infer that DNA is the genetic material. Copyright © 2022 Pearson Education, Inc. All Rights Reserved DNA Controversy • In the early 1900s, incorrectly interpreted staining experiments led to the false conclusion that amounts of DN A in cells change dramatically. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Genes and Protein • From 1910 to the 1940s, most scientists believed that genes were made of protein rather than DNA. • Proteins were thought to be more complex than DNA and thus more likely to be the genetic material. • This idea prevailed until two important lines of evidence confirmed that DNA is the genetic material. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Avery, MacLeod, and McCarty Showed That DNA Is the Genetic Material of Bacteria • Frederick Griffith, studying a pathogenic bacterial strain that caused pneumonia in animals, found two forms of the bacterium. • S-strain caused a fatal infection when introduced into mice. • R-strain or dead S-strain was unable to do so. • A mixture of dead S-strain and R-strain caused fatal infection. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Griffith’s Experiment on Genetic Transformation in Pneumococcus (1 of 2) Figure 16.2 Griffith’s Experiment on Genetic Transformation in Pneumococcus.. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Genetic Transformation • When dead S-strain and living R-strain were mixed and used to infect mice, the mice died. • Griffith found many live S-strain bacteria in the dead mice • He concluded that the R-strain had been converted into Sstrain, by a substance in the S-strain. He called it the transforming principle. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Avery and Colleagues Identified the Transforming Substance • Oswald Avery and colleagues followed up the experiments of Griffith by trying to determine what the transforming substance was. • They fractionated extracts of the S-strain bacteria and found that only the nucleic acid fraction was able to transform the R-strain. • Digesting the DNA from the extract prevented transformation. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Griffith’s Experiment on Genetic Transformation in Pneumococcus (2 of 2) Figure 16.2 Griffith’s Experiment on Genetic Transformation in Pneumococcus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Hershey and Chase Showed That DN A Is the Genetic Material of Viruses • Bacteriophages (or just phages) are viruses that infect bacteria. • Phage T2, T4, and T6 are the best studied. • During infection, the virus attaches to the bacterial cell surface and injects material into the cell. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Structure and Lifecycle of Bacteriophage T4 Figure 16.3 The Structure and Life Cycle of Bacteriophage T4. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Infection by T4 Phage • Once injected into the host cell, the genetic information of the phage is transcribed and translated. • Phage DNA and capsid proteins self-assemble into hundreds of new phage particles. • The infected cell breaks open and releases the new phage particles into the medium. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Virulent and Temperate Phages (1 of 2) • This cycle (injections, transcription, translation, selfassembly, and then cell lyses) is called lytic growth. • Lytic growth is characteristic of a virulent phage. • A temperate phage can either produce lytic growth or integrate its DNA into the bacterial chromosome. • Bacteriophage λ (lambda) is a temperate phage. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Virulent and Temperate Phages (2 of 2) • In the integrated or lysogenic state, the DNA of a temperate phage is called a prophage. • The prophage is replicated with the bacterial DNA. During this time, the phage genes are inactive or repressed. • Under certain conditions, the prophage DNA is excised. This process is called induction. • The phage enters the lytic cycle. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Genetic Material of T2 Phage • Alfred Hershey and Martha Chase labeled phage in two different experiments to distinguish protein from DN A. • They labeled proteins with radioactive sulfur, 35S, and the DNA with radioactive phosphorus, 32P. • In two separate experiments, they allowed the labeled phages to infect bacteria. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Detection of Radioactivity • Once the genetic material is injected into the bacteria, the empty phage protein coats (“ghosts”) were removed by agitating cells in a blender. • Cells were recovered by centrifugation. • They then measured the radioactivity in the supernatant (phage coats) and the pellet (cells at the bottom of the tube). Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Results of the Experiment • The results showed that most of the 32P remained with the bacterial cells, but the majority of the 35S was found in the surrounding medium. • Hershey and Chase concluded that DNA and not protein had been injected into the bacterial cells. • Therefore, DNA was the genetic material of the phage T2. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Hershey-Chase Experiment: DNA as the Genetic Material of Phage T2 Figure 16.4 The Hershey–Chase Experiment: Copyright © 2022 Pearson Education, Inc. All Rights Reserved RNA Is the Genetic Material in Some Viruses • Some types of bacteriophages carry single-stranded DNA as their genetic material, and many carry genetic material as RNA. • The tobacco mosaic virus (TMV) is an example. • An experiment where TMV and another RNA virus Holmes ribgrass (HR) coat proteins and RNA were mixed so that whichever RNA was present determined the type of lesion on the plant. Copyright © 2022 Pearson Education, Inc. All Rights Reserved RNA Is the Genetic Material of Tobacco Mosaic Virus Figure 16.5 R N A Is the Genetic Material of Tobacco Mosaic Virus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved RNA Is the Genetic Material in Some Viruses-Retroviruses • Retroviruses (which are another type of RNA viruses) are important to human health. • HIV (human immunodeficiency virus) is an example. • RNA serves as a template for making complementary DN A in the cell using the enzyme reverse transcriptase. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Retroviruses (1 of 3) • In the virus, two copies of the RNA genome are enclosed within a protein capsid. • Each RNA molecule has an attached copy of reverse transcriptase. • The virus first binds the surface of the host cell, and its envelope fuses with the plasma membrane. • Once inside the cell, the viral reverse transcriptase catalyzes synthesis of a DNA strand complementary to the viral RNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Retroviruses (2 of 3) • The reverse transcriptase then catalyzes the formation of a second DNA strand complementary to the first. • The resulting double-stranded DNA then enters the nucleus and integrates into the genome of the host. • The integrated viral genome is called a provirus. • It is replicated every time the host cell replicates its own D NA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Retroviruses (3 of 3) • Transcription of the proviral DNA produces RNA transcripts that function in two ways. • First, they serve as m RNA molecules that direct synthesis of viral proteins. • Second, some of these same transcripts are packaged with viral proteins into new virus particles. • The new viruses “bud” from the plasma membrane without necessarily killing the infected cell. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Reproductive Cycle of a Retrovirus Figure 16.6 The Reproductive Cycle of a Retrovirus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved RNA Tumor Viruses • Some retroviruses, called RNA tumor viruses, can cause cancer. • One type of these viruses carries a cancer-causing oncogene in its genome. • The oncogene is a mutated version of a normal cellular gene, called a proto-oncogene. • The second type of these viruses does not carry an oncogene, but integrates into a host DNA such that the proto-oncogene is converted into an oncogene. Copyright © 2022 Pearson Education, Inc. All Rights Reserved 16.2 DNA Structure • Once it was determined that DNA was the genetic material, a new set of questions began to emerge. • One of the first was how cells are able to accurately replicate their DNA to be passed on during cell division. • Answering this question required an understanding of the three-dimensional structure of DNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chargaff’s Rules Reveal That A = T and G = C • Erwin Chargaff was interested in the base composition of DNA and used chromatographic methods to separate and quantify the relative amounts of the four bases. • He showed that the DNA from different cells of a given species has the same percentage of each of the four bases. • The base composition varies among species. Copyright © 2022 Pearson Education, Inc. All Rights Reserved DNA Base Composition Data That Led to Chargaff’s Rules Table 16.1 DNA Base Composition Data That Led to Chargaff’s Rules Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chargaff’s Most Striking Observation • Chargaff observed that for all DNA samples examined, the number of A = the number of T, and the number of G = the number of C. • These are called Chargaff’s rules. • The significance was not understood until Watson and Crick proposed the double-helix model for DNA structure. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Watson and Crick Discovered That DNA Is a Double Helix • James Watson and Frances Crick built wire models to try to determine the structure of DNA that agreed with everything known about DNA. • It was known that DNA had a sugar phosphate backbone with nitrogenous bases attached to each sugar. • It was known that at physiological pH, the bases would be able to form hydrogen bonds with each other. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Double Helix Model • The critical evidence came from X-ray diffraction data produced by Rosalind Franklin. • It revealed that DNA was a long thin helical molecule. • Based on this information and other observations, Watson and Crick produced the double helix model. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Modeling DNA Structure (1 of 2) Figure 16.7 Modeling DNA Structure. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Modeling DNA Structure (2 of 2) Figure 16.7 Modeling DNA Structure. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Watson-Crick Model (1 of 2) • In the double helix, the sugar-phosphate backbones are on the outside of the helix with the bases on the inside, forming “steps” in a “spiral staircase.” • There are ten nucleotide pairs per complete turn, and 0.34 nm per nucleotide pair. • The 2-nm diameter of the helix is too small for purines and too large for pyrimidines, but just right for one of each. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Watson-Crick Model (2 of 2) • The purine-pyrimidine pairing is consistent with Chargaff’s rules. • The two strands are held together by hydrogen bonding between bases on opposite strands. • The hydrogen bonds fit within the helix only when they form between adenine and thymine or guanine and cytosine. • The base sequence of one strand determines the sequence of opposing strand; the strands are complementary. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The DNA Double Helix Figure 16.8 The DNA Double Helix. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Replication of Genetic Information • The most important aspect of the double helix model was that it suggested a mechanism for replication of DNA. • The two strands could separate so that each could act as a template to dictate synthesis of a new complementary strand. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Key Features of DNA Structure • The strands of DNA are twisted around each other so that there is a major groove and a minor groove. • The phosphodiester bonds that join the 5′ carbon of one nucleotide to the 3′ carbon of the next are oriented in opposite directions in the two DNA strands. • This is called antiparallel orientation. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Measuring DNA Length • DNA length is measured in base pairs (bp). • Larger stretches are measured in multiples of a single base pair—for example, the kilobase (kb) is 1000 bp and a megabase (Mb) is 1,000,000 bp. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Structural Variants of DNA (1 of 2) • The right-handed helix is called B-DNA. • Naturally occurring B- DNA helices are flexible with variable shapes and dimensions, depending on nucleotide sequence; it is the main form of DNA. • Z-DNA is a left-handed helix; its biological significance is not well understood. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Structural Variants of DNA (2 of 2) • A- DNA is a right-handed helix, shorter and thicker than BDNA. • A-DNA is created artificially; there is very little naturally occurring A- DNA. • However, most RNA double helices are of the A type. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Alternative Forms of DNA Figure 16.9 Alternative Forms of DNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved DNA Can Be Interconverted Between Relaxed and Supercoiled Forms • The DNA double helix can be twisted upon itself to form supercoiled DNA. • Twisted DNA that is twisted even further in the same direction is called a positive supercoil. • Twisting the DNA in the opposite direction that it is already coiled is called a negative supercoil. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Supercoiling • Supercoiling occurs in both linear and circular DNA molecules but is more easily studied in circular DNA. • A DNA molecule can go back and forth between the supercoiled state and the nonsupercoiled, or relaxed, state. • Extensive supercoiling helps make chromosomal DNA more compact. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Interconversion of Relaxed and Supercoiled DNA Figure 16.10 Interconversion of Relaxed and Supercoiled D N A. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Interconversion Between Relaxed and Supercoiled DNA • Topoisomerases can both induce and relax supercoils. • Type I topoisomerases: introduce transient single-strand breaks in DNA. • Type II topoisomerases: introduce double-strand breaks; one example in bacteria is DNA gyrase. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Reactions Catalyzed by Topoisomerases I and II Figure 16.11 Reactions Catalyzed by Topoisomerases I and II. Copyright © 2022 Pearson Education, Inc. All Rights Reserved DNA Gyrase, a Bacterial Type II Topoisomerase • Bacteria have a type II topoisomerase called DNA gyrase, which can induce and relax supercoiling. • It is involved in DNA replication. • It can relax positive supercoiling and induce negative supercoiling. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Two Strands of a DNA Double Helix Can Be Denatured and Renatured • DNA strands are bound together by relatively weak noncovalent bonds. • Strand separation (DNA denaturation or melting) can be induced experimentally by raising temperature or pH. • The reverse process is DNA renaturing (reannealing). • The process can be monitored because single- and double-stranded DNA differ in light absorption. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Denaturation • All DNA absorbs light, with an absorption maximum around 260 nm. • As the strands separate, the absorbance increases rapidly. • The temperature at which half of the absorbance change is reached is called the DNA melting temperature, Tm. • The value of Tm reflects how tightly the DNA double helix is held together. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Influences on DNA Melting Temperature • The GC content, as they are held together by three hydrogen bonds. • A major interaction within each single strand stabilizes the double helix. Base stacking involves interactions between adjacent aromatic rings by hydrophobic and van der Waals interactions. • Proper base pairing. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Thermal Denaturation Profile for D N A Figure 16.12 A Thermal Denaturation Profile for D N A. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Dependence of D N A Thermal Denaturation on Base Composition Figure 16.13 Dependence of D N A Thermal Denaturation on Base Composition. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nucleic Acid Hybridization • Nucleic acid hybridization, a family of procedures for identifying nucleic acids based on sequences to bind (hybridize) to each other. Leads to the formation of DNA-D NA, DNA-RNA, or RNA-RNA hybrids. • Denatured DNA is incubated with a purified singlestranded DNA (a probe) with a sequence complementary to the sequence one is trying to detect. • This is used in the technique fluorescence in situ hybridization, FISH. Copyright © 2022 Pearson Education, Inc. All Rights Reserved DNA Denaturation and Renaturation Figure 16.14 DNA Denaturation and Renaturation. Copyright © 2022 Pearson Education, Inc. All Rights Reserved 16.3 DNA Packaging • Very long molecules of DNA must be fit into the cell and, in the case of eukaryotes, into the nucleus. • DNA packaging is a challenge for all forms of life. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Bacteria Package DNA in Bacterial Chromosomes and Plasmids • Bacterial chromosomes were once thought to be naked DN A. • However, it is now known that the DNA is packaged somewhat similarly to the chromosomes of eukaryotes. • The main bacterial genome is called the bacterial chromosome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Bacterial Chromosomes (1 of 2) • Bacteria have single, multiple, linear, or circular chromosomes depending on the species, but a single circular chromosome is most common. • The DNA molecule is bound to small amounts of protein and localized to a region of the bacterial cell called the nucleoid. • The bacterial DNA is negatively supercoiled and folded into loops. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Bacterial Chromosomes (2 of 2) • The loops of bacterial DNA are held in place by RNAs and proteins. • Treatment with ribonuclease degrades RNA and releases some of the loops. • Nicking with a topoisomerase does not affect the loops but relaxes the supercoils. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Bacterial Nucleoid Figure 16.15 The Bacterial Nucleoid. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Bacterial Plasmids • Besides the chromosome, bacteria may contain one or more plasmids, small, usually circular DNA molecules containing genes for their own replication. • They may also carry genes for cellular functions. • Most are supercoiled, and though they replicate autonomously, replication is somewhat synchronous with the chromosome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Types of Plasmids (1 of 2) • F (fertility) factors are involved in the process of conjugation. • R (resistance) factors carry genes that impart drug resistance to the bacterium. • col (colinogenic) factors allow bacteria to secrete colicins, compounds that kill nearby bacteria that lack the col factor. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Types of Plasmids (2 of 2) • Virulence factors enhance the ability to cause disease by producing toxic proteins. • Metabolic plasmids produce enzymes required for certain metabolic reactions. • Cryptic plasmids have no known function. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Eukaryotes Package DNA in Chromatin and Chromosomes • In eukaryotes, there is more DNA per cell, and it interacts with more proteins. • When bound to proteins, DNA is converted into chromatin. • At the time of division, the chromatin fibers condense into a more compact structure, the chromosome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Histones • Histones are a group of small basic proteins with high lysine and arginine content. • The negatively charged DNA binds stably to the positively charged proteins. • The mass of histones in a chromosome is approximately equal to the mass of the DNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Types of Histones • There are five main types of histones, H1, H2A, H2B, H3, and H4. • Chromatin contains about equal numbers of all of these except H1, which is present in about half the amount of the others. • Chromatin also contains a number of nonhistone proteins, which play a variety of roles. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nucleosomes Are the Basic Unit of Chromatin Structure • In the 1960s, X-ray diffraction studies revealed that chromatin has a repeating structural subunit seen in neither DNA nor histones alone. • When isolated from cells, chromatin fibers appear as a series of tiny particles attached by thin filaments (“beadson-a-string”). • The “beads” and the thin filament connecting them are called nucleosomes. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nucleosomes Figure 16.16 Nucleosomes. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Evidence for Nucleosomes • Chromatin can be exposed to a nuclease that cleaves DN A, and the partially degraded DNA is separated from proteins. • Electrophoresis shows a distinctive pattern of DNA fragments in repeating 200-bp intervals. • This pattern is not generated when DNA alone is digested, and it suggests that nucleosomes occur at 200-bp intervals. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Evidence That Proteins Are Clustered at 200Base-Pair Intervals Along the DNA Molecule in Chromatin Fibers Figure 16.17 Evidence That Proteins Are Clustered at 200-Base-Pair Intervals Along the D N A Molecule in Chromatin Fibers. Copyright © 2022 Pearson Education, Inc. All Rights Reserved A Histone Octamer Forms the Nucleosome Core • Roger Kornberg and colleagues showed that nucleosomes can be assembled in vitro only when the histones used were isolated gently. • Under these isolation procedures, histone dimers H2A– H2B and H3–H4 remained intact. • They concluded that the H2A–H2B and H3–H4 complexes were an integral part of the nucleosome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Investigation of the Nucleosome • Kornberg and colleagues used chemical crosslinking to show that nucleosomes contain an octamer of eight histones. • Histone octamers contained two H2A–H2B dimers and two H3–H4 dimers, with the DNA wrapped around the octamer. • The octomer plus 146 bp of DNA is the core particle; extra DNA from the original 200 bp is called linker DNA. H1 is associated with the linker DNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved More Investigation of the Nucleosome • The amount of linker DNA varies among organisms, but the DNA associated with the core particle always measures close to 146 bp. • This is enough DNA to wrap 1.7 times around the core particle. • Histone H1 is thought to be associated with the linker DN A, found between core particles. Copyright © 2022 Pearson Education, Inc. All Rights Reserved A Closer Look at the Nucleosome Structure Figure 16.18 A Closer Look at Nucleosome Structure. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nucleosomes Are Packed Together to Form Chromatin Fibers and Chromosomes • Nucleosome formation is the first step in packaging of nuclear DNA. • Isolated chromatin (beads on a string) measures about 10 nm in diameter, but chromatin of intact cells measures about 30 nm (the 30-nm chromatin fiber). • Histone H1 facilitates formation of the 30-nm fiber. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Levels of Chromatin Packing Figure 16.19 Levels of Chromatin Packing. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Further Packing of Chromatin • The 30-nm fiber seems to be packed together in an irregular, three-dimensional zigzag structure. • These fibers fold into DNA loops 50,000–100,000 bp in length, stabilized by cohesin protein (in mammals) and CT CF. • The loops are spatially arranged through attachment to nonhistone proteins that form a chromosomal scaffold. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Electron Micrograph Showing the Protein Scaffold That Remains After Removing Histones from Human Chromosomes Figure 16.20 Electron Micrograph Showing the Protein Scaffold That Remains After Removing Histones from Human Chromosomes. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Transcription and Packaging • Transcriptionally active DNA is less tightly packed than inactive DNA. • This makes sense because a “looser” packaging would allow easy access by proteins involved in gene transcription. • The extent to which DNA has been folded can be quantified using the DNA packing ratio. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Changes in Histones and Chromatin Remodeling Proteins Can Alter Chromatin Packing • Cells can tightly regulate the portions of chromatin that are active or inactive, through altering histones. • Each histone has a protruding tail that can be tagged by the addition of methyl, acetyl, phosphate, or other groups. • Various combinations of these tags create a histone code. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Histone Methylation and Acetylation • One tagging reaction is the methylation of lysine via histone methyltransferase. • Methylation can serve as a signal for activation or repression of transcription, depending on the lysine involved. • Acetylation of histone side chains is accomplished by histone acetyltransferases (HATs). • The opposite function is catalyzed by histone deacetylase (HDAC). Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chromatin Remodeling (1 of 2) • Chromatin is referred to as “open” (active) or “closed” (inactive). • Proteins with chromodomains are associated with methylated histones characteristic of “closed” chromatin. • Proteins with bromodomains bind to acetylated DNA associated with “open” chromatin. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chromatin Remodeling (2 of 2) • Other proteins, chromatin remodeling proteins, alter the position of nucleosomes along DNA. • One important class of remodelers is the SWI/SNF family. • These slide nucleosomes or remove them from a region of chromatin, making the DNA more accessible. • The chromatin remodeling can be inherited by daughter cells and is called epigenetics. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Effects of Histone Modification and Chromatin Remodeling Proteins on Chromatin Packing Figure 16.21 Effects of Histone Modification and Chromatin Remodeling Proteins on Chromatin Packing. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chromosomal DNA Contains Euchromatin and Heterochromatin • Sections of chromatin so highly compacted that they show up as dark spots in micrographs are called heterochromatin. • More loosely packed, diffuse chromatin is called euchromatin. • Much of chromatin in metabolically active cells is euchromatic, but in preparation for cell division all the chromatin becomes highly compacted. • After replication, each chromosome is composed of two identical chromatids. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Some Heterochromatin Plays a Structural Role in Chromosomes • Facultative heterochromatin can be converted to euchromatin, and vice versa. • Some heterochromatin is permanently compacted; known as constitutive heterochromatin, it serves structural functions within chromosomes. • Two important types of constitutive heterochromatin are centromeres and telomeres. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Centromeres and Telomeres Are Structural Elements of Chromosomes Figure 16.22 Centromeres and Telomeres Are Structural Elements of Chromosomes. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Centromeres • Centromeres appear as constriction of chromosomes. • Centromere DNA is bound by a complex of proteins and serves important functions. • Centromeres maintain sister chromatid cohesion during mitosis and meiosis. • They also serve as sites of kinetochores, crucial for attaching spindle microtubules to chromosomes during meiosis and mitosis. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Centromere Sequences • Centromeres are characterized by highly repetitive DNA sequences (CEN sequences). • Eukaryotes have their own CEN regions, which are not very similar from one organism to the next. • Centromeric chromatin uses a histone H3 variant called C ENP-A. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Telomeres • Telomeres are found at the tips of chromosomes. • They contain highly repetitive DNA sequences. • Telomeres protect chromosome ends from degradation during each round of DNA replication. • All vertebrates studied so far have the same repeat sequence (TTAGGG). • Telomeres recruit proteins that are involved in structural protection of chromosome tips. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chromosomes Can Be Identified by Unique Banding Patterns • Mitotic chromosomes viewed under the light microscope can be distinguished by size and position of the centromere. • Similar-sized chromosomes can be distinguished by their banding patterns in response to stains. • A common stain is Giemsa, which causes light- and darkstaining chromosome bands called G bands. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Mitotic Human Chromosomes from Metaphase-Arrested Cells Have Unique GBanding Patterns Figure 16.23 Mitotic Human Chromosomes from Metaphase-Arrested Cells Have Unique G-Banding Patterns. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Eukaryotic Chromosomes Contain Large Amounts of Repeated DNA Sequences • In the 1960s, Roy Britten and David Kohne discovered repeat DNA sequences. • They broke DNA into small fragments, denatured them by heating, and allowed them to renature. • The rate of renaturation depends on the concentration of each kind of DNA sequence—those found in high concentration reanneal more quickly. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Bacterial versus Mammalian DNA • When mammalian and bacterial DNA were tested, it was expected that bacterial DNA, having fewer types of DNA sequences, should reanneal much faster. • The results were not as expected; the calf DNA consisted of two classes of sequences that renature at very different rates. • About 40% of the calf DNA renatures more rapidly than bacterial DN A. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Repeated DNA Sequences • The more rapidly annealing sequences of the calf DNA contain repeated DNA sequences that are present in multiple copies. • Eukaryotes have variable amounts of repeated DNA in their genomes; the rest is nonrepeated DNA. • There are two categories of repeated DNA: tandemly repeated DNA and interspersed repeated DNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Renaturation of Calf and E. coli D N A Shows Eukaryotes Have Repetitive D N A Figure 16.24 Renaturation of Calf and E. coli DNA Shows Copyright © 2022 Pearson Education, Inc. All Rights Reserved Categories of Repeated Sequences in Eukaryotic DNA Table 16.2 Categories of Repeated Sequences in Eukaryotic DNA Copyright © 2022 Pearson Education, Inc. All Rights Reserved Tandemly Repeated DNA • One major category of DNA repeats is called tandemly repeated DNA. • The multiple copies are arranged next to each other in a row. • It accounts for 10–15% of a typical mammalian genome; a repeat unit can measure anywhere from 1 to 2000 bp, most of the time less than 10 bases. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Simple-Sequence Repeats • The tandem repeats that are less than 10 bases per repeat comprise a subcategory called simple-sequence repeated DNA. • There can be as many as several hundred thousand copies at selected sites in the genome. • It was originally called satellite DNA. • Typical satellite DNA range usually from 105 to 107 bp in overall length. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Types of Repeat Sequences • The amount of satellite DNA at any given site can vary enormously; typically it ranges from 105 to 107 bp in overall length. • Variable number tandem repeats (VNTRs) refer to short repeats. • Minisatellites are short, 102 to 105 bp in length. • Microsatellites (or short tandem repeats, STRs) are even shorter, 10–100 bp in length, but with numerous sites in the genome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Interspersed Repeated DNA • Interspersed repeated DNAs are scattered around the genome. • Single repeats are hundreds or thousands of bases in length, and the dispersed copies, numbering in hundreds of thousands of copies, are similar but not identical to one another. • They account for 25–50% of mammalian genomes. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Types of Interspersed Repeated DNA • Most interspersed repeated DNA consists of families of transposable elements (transposons), which can move around the genome and leave copies of themselves behind. • Roughly half of the human genome consists of these mobile elements. • The most abundant are called LINEs (long interspersed nuclear elements) and account for about 20% of the genome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved LINEs and SINEs • LINEs are 6000–8000 bp long and contain genes required for their own mobilization. • SINEs are short interspersed nuclear elements and are less than 500 bp. • These rely on enzymes from other elements for their movement. • The most common SINEs in humans are Alu sequences, which account for 10% of the human genome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Types of DNA in the Human Genome Figure 16.25 Types of DNA in the Human Genome. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Eukaryotes Package Some of Their D NA in Mitochondria and Chloroplasts • Mitochondria and chloroplasts have their own chromosomes, which are devoid of histones and are usually circular. • Both organelles can encode some of their own polypeptides but depend on the nuclear genome to encode the rest of them. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Mitochondrial DNA Figure 16.26 Mitochondrial DNA. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Human Mitochondrion • The genome of the human mitochondrion has been sequenced. • It is 16,569 base pairs long and encodes 37 genes, about 5% of all the RNAs and proteins needed by the mitochondrion. • Although its only 5% of the RNAs and proteins needed, it is vital. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Genomic Makeup of the Human Mitochondrion Figure 16.27 Genomic Makeup of the Human Mitochondrion. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Mitochondrial Genomes • The size of mitochondrial genomes varies considerably among organisms. • Yeasts have mitochondrial genomes about five times larger than those of mammals, but most of the extra DNA is noncoding. • A 648-nucleotide sequence sometimes called the DNA bar code can be used to distinguish closely related species. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chloroplast Genomes • Chloroplasts usually possess circular DNA molecules of about 120,000 bp in length, containing around 120 genes. • Subunits of some multimeric protein complexes are encoded by the nuclear genome; this is true for both chloroplasts and mitochondria. Copyright © 2022 Pearson Education, Inc. All Rights Reserved 16.4 The Nucleus • The nucleus is the site within the eukaryotic cell where the chromosomes are localized and replicated and the DNA they contain is transcribed. • It is one of the most prominent and distinguishing features of eukaryotic cells. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Nucleus Figure 16.28 The Nucleus. The nucleus is a prominent structural feature in most eukaryotic cells. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Structural Organization of the Nucleus and Nuclear Envelope Figure 16.29 Structural Organization of the Nucleus and Nuclear Envelope. Copyright © 2022 Pearson Education, Inc. All Rights Reserved A Double-Membrane Nuclear Envelope Surrounds the Nucleus • The nucleus is bounded by a nuclear envelope with an inner and an outer membrane separated by a perinuclear space. • The outer membrane is continuous with the ER and contains proteins that bind actin and intermediate filaments (IFs) of the cytoskeleton. • Tubular invaginations of the envelope project into the nucleus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Pores (1 of 2) • Nuclear pores are specialized channels in the nuclear envelope where inner and outer membranes are fused. • They provide direct contact between the cytosol and the nucleoplasm (interior nuclear space). • They are lined with a protein structure called the nuclear pore complex (NPC). Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Pores (2 of 2) Figure 16.30 Nuclear Pores. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Pore Complex • The NPC is built from about 30 different proteins called nucleoporins. • The complex has a striking octagonal symmetry. • The “central granule” is called the transporter and is likely involved in moving molecules across the nuclear envelope. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Structure of the Nuclear Pore Figure 16.31 Structure of the Nuclear Pore. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Molecules Enter and Exit the Nucleus Through Nuclear Pores • Enzymes and proteins needed in the nucleus must be imported from the cytoplasm. • RNAs that need to be translated and components of ribosomes must be exported from the nucleus. • In addition to all the traffic through the pores, they also mediate passage of small particles, molecules, and ions. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Macromolecular Transport into and out of the Nucleus Figure 16.32 Macromolecular Transport into and out of the Nucleus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Simple Diffusion of Small Molecules Through Nuclear Pores • Small particles, less than 10 nm in diameter, pass through pores at a rate proportional to the size of the particle. • The NPC contains tiny aqueous diffusion channels through which small particles freely move. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Active Transport of Large Proteins and RNA Through Nuclear Pores • Some proteins needed in the nucleus are too large to easily diffuse through the nuclear pores. • These large particles are actively transported across the membrane. • Nuclear localization signals (NLS) enable the protein to be recognized and transported by the nuclear pore complex. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Localization Signals • An NLS is usually 8–30 amino acids in length and often contains proline and the basic amino acids lysine and arginine. • One of the first NSL sequences to be characterized was part of the large T antigen, a protein made by simian virus 40 (SV40). • It is a stretch of seven amino acids near the C-terminus of the protein. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Importance of Nuclear Localization Signals for Nuclear Import (1 of 2) Figure 16.33 The Importance of Nuclear Localization Signals for Nuclear Import. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Importance of Nuclear Localization Signals for Nuclear Import (2 of 2) Figure 16.33 The Importance of Nuclear Localization Signals for Nuclear Import. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Import via the Ran/Importin Pathway (1 of 2) 1. A cytoplasmic protein with an NLS is recognized by a receptor protein called an importin, which binds the NLS and mediates movement of the protein to a nuclear pore. 2. The importin-protein complex is transported into the nucleus by the transporter at the center of the NPC. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Import via the Ran/Importin Pathway (2 of 2) 3. Inside the nucleus, the importin associates with a GTPbinding protein called Ran, causing importin to release the NLS-containing protein (3). 4. The Ran-G TP importin complex is transported back to the cytoplasm through the NPC (4). 5. In the cytoplasm, the importin is released as GTP is hydrolyzed (5). Copyright © 2022 Pearson Education, Inc. All Rights Reserved Transport Through the Nuclear Pore Complex Figure 16.34 Transport Through the Nuclear Pore Complex. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Export via Ran-Independent and Ran-Dependent Pathways (1 of 2) • Export occurs by a process comparable to import. • Transport out of the nucleus is used mainly for RNA molecules. • Some traffic out of the nucleus does not appear to require Ran, for instance mRNAs. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Export via Ran-Independent and Ran-Dependent Pathways (2 of 2) • RNA export is mediated by adaptor proteins that bind to the RNA. • The adaptor proteins contain sequences called nuclear export signals (NES), which target the proteins—and the bound RNAs—for export. • NES sequences are recognized by exportins, which mediate transport of the complexes out of the nucleus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Maintaining a Ran-GTP Gradient Across the Membrane • Ran-G TP is maintained at high levels inside the nucleus by a guanine-nucleotide exchange factor (GEF) that promotes Ran to bind GTP. • The cytosol contains a GTPase activating protein (GAP) that promotes hydrolysis of GTP by Ran. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Function of the Ran-GTP Gradient Across the Membrane • The high nuclear Ran-GTP promotes the release of NLScontaining cargo from importin. • It also promotes the binding of NES-containing cargo to exportin. • Nuclear transport factor 2 (NTF2) shuttles Ran-GDP back into the nucleus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Nucleus Is Mechanically Integrated with the Rest of the Cell • The nuclear matrix (nucleoskeleton) is an insoluble fibrous network that helps maintain the shape of the nucleus. • The existence of a nucleoskeleton is debated. • Not debated, is the connections between the nuclear membrane and the cell’s cytoskeleton. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Nuclear Matrix and the Nuclear Lamina (1 of 2) Figure 16.35 The Nuclear Matrix and the Nuclear Lamina. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Nuclear Matrix and the Nuclear Lamina (2 of 2) Figure 16.35 The Nuclear Matrix and the Nuclear Lamina. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Nuclear Lamina • The inner nuclear membrane is connected by several proteins to the outer nuclear membrane, which is connected to the cytoskeleton. • The nuclear lamina is a thin dense meshwork of fibers lining the inner surface of the inner nuclear membrane. • It is made of intermediate filaments made from lamins. • Plants and fungi do not have lamins. It is presumed they rely on different proteins for nuclear structure. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Chromatin Is Located Within the Nucleus in a Nonrandom Fashion • Most of the time, a cell’s chromatin fibers are extended and dispersed through the nucleus. • The chromatin of each chromosome has its own discrete location (chromosome territory). • In situ hybridization, using nucleic acid probes specific for sequences specific to individual chromosomes, demonstrates this. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Nucleolus Is Involved in Ribosome Formation • The nucleolus is the place in the nucleus where ribosomal subunits are assembled. • There can be more than one nucleoli in a cell. • Fibrils in the nucleolus contain DNA that is being transcribed into ribosomal RNA (rRNA). • Granules in the nucleolus are rRNA molecules being packaged with proteins. Copyright © 2022 Pearson Education, Inc. All Rights Reserved The Nucleolus Figure 16.36 The Nucleolus. Copyright © 2022 Pearson Education, Inc. All Rights Reserved Copyright This work is protected by United States copyright laws and is provided solely for the use of instructors in teaching their courses and assessing student learning. Dissemination or sale of any part of this work (including on the World Wide Web) will destroy the integrity of the work and is not permitted. The work and materials from it should never be made available to students except by instructors using the accompanying text in their classes. All recipients of this work are expected to abide by these restrictions and to honor the intended pedagogical purposes and the needs of other instructors who rely on these materials. Copyright © 2022 Pearson Education, Inc. All Rights Reserved