Lecture 11 Molecular Structure of DNA and RNA and Transposable elements.pdf

MOLECULAR STRUCTURE OF DNA AND RNA ©Scott Camazine/123RF INTRODUCTION In this chapter we will shift our attention to molecular genetics, which is the study of DNA structure and function at the molecular level Dramatic a...

MOLECULAR STRUCTURE OF DNA AND RNA ©Scott Camazine/123RF INTRODUCTION In this chapter we will shift our attention to molecular genetics, which is the study of DNA structure and function at the molecular level Dramatic advances in techniques and approaches have greatly expanded our understanding of molecular genetics, transmission genetics, and population genetics To a large extent, our knowledge of genetics comes from our knowledge of the molecular structure of DNA and RNA 9.1 IDENTIFICATION OF DNA AS THE GENETIC MATERIAL 1 To fulfill its role, the genetic material must meet several criteria Information: It must contain the information necessary to make an entire organism Transmission: It must be passed from parent to offspring Replication: It must be copied in order to be passed from parent to offspring Variation: It must be capable of changes to account for the known phenotypic variation in each species 9.1 IDENTIFICATION OF DNA AS THE GENETIC MATERIAL 2 The data of many geneticists, including Mendel, were consistent with these four properties However, the chemical nature of the genetic material cannot be identified solely by genetic crosses The identification of DNA as the genetic material involved a series of different experimental approaches FREDERICK GRIFFITH EXPERIMENTS WITH STREPTOCOCCUS PNEUMONIAE Griffith studied a bacterium (pneumococci) now known as Streptococcus pneumoniae S. pneumoniae comes in two strains Type S  Smooth Secrete a polysaccharide capsule Protects bacterium from the immune system of animals Produce smooth colonies on solid media Type R  Rough Unable to secrete a capsule Produce colonies with a rough appearance GRIFFITH’S EXPERIMENTS ON GENETIC TRANSFORMATION 1 In 1928, Griffith conducted experiments using two strains of S. pneumoniae: type S and type R 1. Inject mouse with live type S bacteria Mouse died & type S bacteria recovered from the mouse’s blood 2. Inject mouse with live type R bacteria Mouse survived & no living bacteria isolated from the mouse’s blood GRIFFITH’S EXPERIMENTS ON GENETIC TRANSFORMATION 2 3. Inject mouse with heat-killed type S bacteria Mouse survived & no living bacteria isolated from the mouse’s blood 4. Inject mouse with live type R + heat-killed type S cells Mouse died & type S bacteria recovered from the mouse’s blood GRIFFITH’S EXPERIMENTS ON GENETIC TRANSFORMATION 3 (a) Live type (b) Live type R S Access the text alternative for slide images. GRIFFITH’S EXPERIMENTS ON GENETIC TRANSFORMATION 4 (c) Dead type S (d) Live type R + dead type S Access the text alternative for slide images. TRANSFORMING PRINCIPLE 1 Griffith concluded that something from the dead type S bacteria was transforming type R bacteria into type S He called this process transformation The substance that allowed this to happen was termed the transforming principle Griffith did not know what type of substance it was TRANSFORMING PRINCIPLE 2 The formation of a capsule by these bacteria fulfilled the 4 required properties of a “genetic material” Transformed bacteria acquired information to make the capsule Variation exists in ability to make a capsule The information required to create a capsule is replicated and transmitted from mother to daughter cells The nature of the transforming principle was subsequently determined using experimental approaches that incorporated various biochemical techniques THE EXPERIMENTS OF AVERY, MACLEOD AND MCCARTY 1 Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material They carried out their experiments in the 1940s At that time, it was known that DNA, RNA, proteins and carbohydrates are the major constituents of living cells THE EXPERIMENTS OF AVERY, MACLEOD AND MCCARTY 2 They prepared cell extracts from type S cells and purified each type of macromolecule Only the extract that contained purified DNA was able to convert type R bacteria into type S Treatment of the DNA extract with RNase or protease did not eliminate transformation; treatment with DNase did THE EXPERIMENTS OF AVERY, MACLEOD AND MCCARTY 3 Access the text alternative for slide images. EVIDENCE PROVIDED BY HERSHEY AND CHASE 1 Hershey and Chase provided evidence that DNA is the genetic material of T2 phage Used radioisotopes to distinguish DNA from proteins 32P labels DNA specifically 35S labels proteins specifically Radiolabeled phages were used to infect non-radioactive Escherichia coli cells EVIDENCE PROVIDED BY HERSHEY AND CHASE 2 After allowing sufficient time for infection to proceed, the residual phage particles were sheared off the cells Most of the 32P had entered the bacterial cells (DNA) Most of the 35S remained outside the cells (protein) Indicates that DNA is the genetic material 9.2 OVERVIEW OF DNA AND RNA STRUCTURE 1 DNA (deoxyribonucleic acid), and its molecular cousin RNA (ribonucleic acid), are known as nucleic acids First identified by Friedrich Miescher in 1869 in waste surgical bandages Named the substance “nuclein” Material from the nucleus of a cell Later research showed that DNA (and RNA) release H+ in water and therefore are acids Became named “nucleic acids” 9.2 OVERVIEW OF DNA AND RNA STRUCTURE 2 DNA and RNA are large macromolecules with several levels of complexity Nucleotides form the repeating unit of nucleic acids Nucleotides are linked to form a linear strand of RNA or DNA In DNA, two strands can interact to form a double helix The 3-D structure of DNA results from folding and bending of the double helix. Interaction of DNA with proteins produces chromosomes within living cells FIGURE 9.3 Nucleotide s Single strand Double helix Three-dimensional structure 9.3 NUCLEOTIDE STRUCTURE The nucleotide is the repeating structural unit of DNA and RNA A nucleotide has three components A phosphate group A pentose sugar Ribose in RNA Deoxyribose in DNA A nitrogenous (nitrogen-containing) base THE COMPONENTS OF NUCLEOTIDES Access the text alternative for slide images. FIGURE 9.5 (a) Repeating unit of (b) Repeating unit of deoxyribonucleic acid (DNA) ribonucleic acid (RNA) TERMINOLOGY OF NUCLEIC ACID UNITS 1 Base + sugar is a nucleoside Examples Adenine + ribose = Adenosine Adenine + deoxyribose = Deoxyadenosine Base + sugar + phosphate(s) is a nucleotide Examples Adenosine monophosphate (AMP) Adenosine diphosphate (ADP) Adenosine triphosphate (ATP) TERMINOLOGY OF NUCLEIC ACID UNITS 2 9.4 STRUCTURE OF A DNA STRAND In a DNA strand, nucleotides are linked together by covalent bonds (called ester bonds) A phosphate connects the 5’ carbon of one nucleotide to the 3’ carbon of an adjacent nucleotide; this is called a phosphodiester linkage A DNA strand has 5’ to 3’directionality In a strand, all sugar molecules are oriented in the same direction The phosphates and sugar molecules form the backbone of the nucleic acid strand The bases project from the backbone NUCLEOTIDES ARE LINKED IN STRANDS Access the text alternative for slide images. 9.5 DISCOVERY OF THE DOUBLE HELIX In 1953, James Watson and Francis Crick elucidated the double helical structure of DNA The scientific framework for their breakthrough was provided by other scientists including Linus Pauling Rosalind Franklin and Maurice Wilkins Erwin Chargaff LINUS PAULING AND THE Α HELIX In the early 1950s, Pauling proposed that regions of protein can fold into a secondary structure called an α-helix To elucidate this structure, he built ball-and-stick models (a) An α helix in a protein Access the text alternative for slide images. ROSALIND FRANKLIN PERFORMED X-RAY DIFFRACTION OF DNA FIBERS 1 Working in the same laboratory as Maurice Wilkins, Rosalind Franklin used X-ray diffraction to study wet fibers of DNA A diffraction pattern is interpreted (using mathematical theory) to provide information concerning the structure of a molecule ROSALIND FRANKLIN PERFORMED X-RAY DIFFRACTION OF DNA FIBERS 2 (b) X-ray diffraction of wet DNA fibers ROSALIND FRANKLIN PERFORMED X-RAY DIFFRACTION OF DNA FIBERS 3 Franklin made marked advances in X-ray diffraction techniques with DNA The diffraction pattern she obtained suggested several structural features of DNA Helical More than one strand 10 base pairs per complete turn These findings were instrumental in solving the structure of DNA; her results were shared with Watson and Crick, presumably without her knowledge ERWIN CHARGAFF’S EXPERIMENT Chargaff pioneered many of the biochemical techniques for the isolation, purification and measurement of nucleic acids from living cells It was known that DNA contained the four bases: A, G, C and T Chargaff analyzed the base composition of DNA isolated from many different species THE GOAL: DISCOVERY-BASED SCIENCE An analysis of the base composition of DNA in different species may reveal important features about the structure of DNA CHARGAFF’S EXPERIMENTAL PROTOCOL 1 1. For each type of cell, extract the chromosomal material. This can be done in a variety of ways, including the use of high salt, detergent, or mild alkali treatment. Note: The chromosomes contain both DNA and protein. 2. Remove the protein. This can be done in several ways, including treatment with protease. 3. Hydrolyze the DNA to release the bases from the DNA strands. A common way to do this is by strong acid treatment. CHARGAFF’S EXPERIMENTAL PROTOCOL 2 4. Separate the bases by chromatography. Paper chromatography provides an easy way to separate the four type of bases. (The technique of chromatography is described in Appendix A.) 5. Extract bands from paper into solutions and determine the amounts of each base by spectroscopy. Each base will absorb light at a particular wavelength. By examining the absorption profile of a sample of base, it is then possible to calculate the amount of base. (Spectroscopy is described in Appendix A.) 6. Compare the base content in the DNA from different organisms. CHARGAFF’S EXPERIMENTAL PROTOCOL 3 Access the text alternative for slide images. THE DATA Base Content in the DNA from a variety of Organisms* Percentage of Base Content (Based on Molarity) Organism Adenine Thymine Guanine Cytosine E. coli 26.0 23.9 24.9 25.2 S. pneumoniae 29.8 31.6 20.5 18.0 Yeast 31.7 32.6 18.3 17.4 Turtle red blood cells 28.7 27.9 22.0 21.3 Salmon sperm 29.7 29.1 20.8 20.4 Chicken red blood cells 28.0 28.4 22.0 21.6 Human liver cells 30.3 30.3 19.5 19.9 *When the base compositions from different tissues within the same species were measured, similar results were obtained. These data were compiled from several sources. See E. Chargaff and J. Davidson, Eds. (1995), The Nucleic Acids. Academic Press, New York. INTERPRETING THE DATA Hundreds of measurements were made, and the compelling observation was that Percent of adenine = percent of thymine Percent of cytosine = percent of guanine This observation became known as Chargaff ’s rule It was a crucial piece of evidence that Watson and Crick used to elucidate the structure of DNA WATSON AND CRICK 1 Watson and Crick set out to solve the structure of DNA They tried to build ball-and-stick models that incorporated all known experimental observations Sugar-phosphate backbone on the outside Bases projecting toward each other They first considered a structure in which bases form H bonds with identical bases in the opposite strand, but later realized this was incorrect For example, A to A, T to T, C to C, and G to G WATSON AND CRICK 2 Watson & Crick later realized that the hydrogen bonding of A to T was structurally similar to that of C to G, prompting further modeling with AT and CG interactions between the two DNA strands Their final double helical model was consistent with all known data about DNA structure Watson, Crick and Maurice Wilkins were awarded the Nobel Prize in 1962 Rosalind Franklin died in 1958, and Nobel prizes are not awarded posthumously FIGURE 9.11 9.6 STRUCTURE OF THE DNA DOUBLE HELIX 1 Two strands are twisted together around a common axis There are 10 base pairs (bp) and 3.4 nm per complete turn of the helix The two strands are antiparallel One runs in the 5’ to 3’ direction and the other 3’ to 5’ The helix is right-handed As it spirals away from you, the helix turns in a clockwise direction 9.6 STRUCTURE OF THE DNA DOUBLE HELIX 2 Key Features Two strands of DNA form a right- handed double helix. The bases in opposite strands hydrogen bond according to the AT/GC rule. The 2 strands are antiparallel with regard to their 5’ to 3’ directionality. There are ~10.0 nucleotides in each strand per complete 360° STABILIZATION OF THE DOUBLE HELIX 1 The double-helical structure of DNA is stabilized by: Hydrogen bonding between complementary bases A bonded to T by two hydrogen bonds C bonded to G by three hydrogen bonds Base stacking Within the DNA, the bases are oriented so that the flattened regions are facing each other STABILIZATION OF THE DOUBLE HELIX 2 GROOVES ON THE DNA DOUBLE HELIX 1 There are two asymmetrical grooves on the outside of the helix Major groove Minor groove Certain proteins can bind within these grooves They can thus interact with a particular sequence of bases GROOVES ON THE DNA DOUBLE HELIX 2 (a) Ball-and-stick model of (b) Space-filling model of DNA DNA DNA FORMS ALTERNATIVE TYPES OF DOUBLE HELICES The DNA double helix can form different types of secondary structure The predominant form found in living cells is B DNA Under certain conditions, Z DNA double helices can form Z DNA Left-handed helix 12 bp per turn Its formation is favored by Alternating purine/pyrimidine sequences, at high salt concentrations (for example GCGCGCGCGC) Cytosine methylation, at low salt concentrations Negative supercoiling May play a role in transcription and chromosome structure Recognized by cellular proteins May alter chromosome compaction COMPARISON OF B DNA AND Z DNA 1 B DNA Bases relatively perpendicular to the central axis Z DNA Bases substantially tilted relative to the central axis Sugar-phosphate backbone follows a zigzag pattern COMPARISON OF B DNA AND Z DNA 2 (a) Molecular Structures (b) Space-filling models Illustration, Irving Geis. Image from the Irving Geis Collection, Howard Hughes Medical Institute. Rights owned by HHMI. Not to be reproduced without permission 9.7 RNA STRUCTURE 1 The primary structure of an RNA strand is much like that of a DNA strand, with a couple of exceptions: RNA uses Uracil as a base, instead of Thymine RNA uses Ribose with 2’ OH, instead of Deoxyribose RNA strands are typically several hundred to several thousand nucleotides in length In RNA synthesis, only one of the two strands of DNA is used as a template 9.7 RNA STRUCTURE 2 Access the text alternative for slide images. STRUCTURE OF RNA MOLECULES 1 Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary base- pairing A to U and C to G Allows short regions to form a double helix RNA double helices typically Are right-handed Have 11 to 12 base pairs per turn Different types of RNA secondary structures are possible STRUCTURE OF RNA MOLECULES 2 (a) Bulge (b) Internal (c) Multibranched (d) Stem loop loop loop loop STRUCTURE OF A TRANSFER RNA Many factors contribute to the tertiary structure of RNA Base-pairing and base stacking within the RNA itself Interactions with ions, small molecules and large proteins (a) Ribbon Model Access the text alternative for slide images. 10.5 TRANSPOSITION Transposition involves the integration of small segments of DNA into a new location in the genome This can occur at many different locations within the genome These small, mobile DNA segments are termed transposable elements (TEs) Sometimes referred to as “jumping genes” TEs were first identified by Barbara McClintock in the early 1950s from her classical studies in corn Since then, many different types of TEs have been found in species as diverse as bacteria, fungi, plants and animals MCCLINTOCK DISCOVERS MOVING LOCI IN CORN 1 During her long career, McClintock identified many unusual features of corn chromosomes She noticed that in one strain of corn, chromosome 9 tended to break at a fairly high rate at the same site McClintock termed this a mutable site or mutable locus Mutable sites are actually locations where transposable elements have been inserted into the chromosomes The mutable locus was named Ds (for dissociation), because chromosomal breakage occurred frequently there MCCLINTOCK DISCOVERS MOVING LOCI IN CORN 2 In one case, Ds was in the middle of a gene affecting kernel color. The C allele provides dark red color, whereas c is a recessive allele of the same gene and causes a colorless kernel. The endosperm of corn kernels is triploid Phenotype : Colorless with red sectors MCCLINTOCK DISCOVERS MOVING LOCI IN CORN 3 She proposed the following: 1. The colorless background of a kernel was due to the transposition of Ds into the C allele, which would inactivate that allele. 2. In a few cells, Ds occasionally transposed out of the C allele during kernel growth, the two parts of the C allele were rejoined, thereby restoring its function. As the kernel grew, such a cell would continue to divide, resulting in a red sector. MCCLINTOCK DISCOVERS MOVING LOCI IN CORN 4 MCCLINTOCK’S WORK WAS NOT INITIALLY ACCEPTED When McClintock published these results in 1951, they were met with great skepticism Most geneticists believed that DNA was stable and permanent and not susceptible to rearrangement Over the next several decades, the scientific community realized that TEs are a widespread phenomenon McClintock was awarded the Nobel Prize in 1983 More than 30 years after her original discovery! DIFFERENT TRANSPOSITION OF TRANSPOSABLE ELEMENTS Many transposable elements have been found in bacteria, fungi, plant and animal cells Transposable elements move by different transposition pathways Two general types of transposition pathways have been identified Simple transposition – TE moves to a new target site Retrotransposition – TE moves via an RNA intermediate FIGURE 10.10 (a) Simple transposition (b) Retrotransposition INSERTION ELEMENTS AND SIMPLE TRANSPOSONS 1 All TEs are flanked by direct repeats (DRs), which are identical base sequences that are oriented in the same direction and repeated Simplest TE is an insertion element; which is flanked by inverted repeats Inverted repeats are DNA sequences that are identical (or very similar) but run in opposite directions Range from 9-40 bp May contain a gene for the enzyme transposase, which catalyzes the transposition event INSERTION ELEMENTS AND SIMPLE TRANSPOSONS 2 Simple transposon carries one or more genes not required for transposition Fig 10.11a (a) Elements that move by simple transposition LTR RETROTRANSPOSONS LTR retrotransposons are evolutionarily related to known retroviruses Retain the ability to move around the genome, though, in most cases, they do not produce mature viral particles Contain long terminal repeats (LTRs) at both ends Typically a few hundred base pairs in length Encode virally related proteins, such as reverse transcriptase and integrase, that are needed for the retrotransposition process NON-LTR RETROTRANSPOSONS Non-LTR retrotransposons do not resemble retroviruses in having LTR sequences May contain a gene that encodes a protein that functions as both a reverse transcriptase and an endonuclease Some non-LTR retrotransposons are evolutionarily derived from normal eukaryotic genes The Alu family of repetitive sequences found in humans is derived from a single ancestral gene known as the 7SL RNA gene This gene sequence has been copied by retrotransposition many times, and the current number of copies is approximately 1 FIGURE 10.11B (b) Elements that move by retrotransposition (via an RNA intermediate) AUTONOMOUS VERSUS NON-AUTONOMOUS ELEMENTS 1 Transposable elements are considered to be complete, or autonomous elements, when they contain all of the information necessary for transposition or retrotransposition A nonautonomous element lacks a gene such as one that encodes transposase or reverse transcriptase, which is necessary for transposition Example: The Ds locus in corn lacks a transposase gene AUTONOMOUS VERSUS NON-AUTONOMOUS ELEMENTS 2 The Ac element (activator element) provides a transposase gene that enables Ds to transpose. Nonautonomous TEs such as Ds can transpose only when Ac is present at another region in the genome TRANSPOSASE CATALYZES THE EXCISION AND INSERTION OF TES The enzyme transposase catalyzes the removal of a TE and its reinsertion at another location Transposase recognizes the inverted repeats at the ends of a TE and brings them close together FIGURE 10.12A (a) Movement of transposon via transposase FIGURE 10.12B (b) The formation of direct repeats SIMPLE TRANSPOSITION CAN INCREASE COPY NUMBER Transposition occurs after the replication fork has passed through the TE, so there are two copies of the TE One of these TEs can transpose ahead of the fork where it is copied again One chromosome will still have one TE, but the other will now have two copies FIGURE 10.13 The bottom copy of DNA has 2 TEs. RETROTRANSPOSONS USE REVERSE TRANSCRIPTASE FOR RETROTRANSPOSITION Retrotransposons use an RNA intermediate in their transposition mechanism LTR retrotransposon movement requires two key enzymes: Reverse transcriptase Integrase FIGURE 10.14 TARGET-SITE PRIMED REVERSE TRANSCRIPTION Non-LTR retrotransposons move by target-site primed reverse transcription Retrotransposon transcribed into RNA with a 3’ polyA tail Target DNA recognized by endonuclease PolyA tail binds to nicked site Reverse transcriptase uses target DNA of the primer and makes a DNA copy of the RNA FIGURE 10.15 (1) FIGURE 10.15 (2) TRANSPOSABLE ELEMENTS INFLUENCES ON MUTATION AND EVOLUTION Over the past few decades, researchers have found that transposable elements probably occur in the genomes of all species Table 10.1 describes several TEs that have been studied in great detail (see your textbook) Table 10.2 describes the relative abundance of TEs in selected species GENOMES OF SELECTED SPECIES TABLE 10.2 Abundance of transposable elements in the genomes of selected species Percentage of the Total Genome Species Composed of TEs* Frog (Xenopus laevis) 77 Corn (Zea mays) 60 Human (Homo sapiens) 45 Mouse (Mus musculus) 40 Fruit fly (Drosophila melanogaster) 20 Nematode (Caenorhabditis elegans) 12 Yeast (Saccharomyces cerevisiae) 4 Bacterium (Escherichia coli ) 0.3 *In some cases, the abundance of TEs may vary somewhat among different strains of the same species. The values reported here are typical values. REPETITIVE SEQUENCES IN EUKARYOTIC GENOMES Some repetitive sequences in eukaryotic genomes are due to the proliferation of TEs In mammals, for example LINEs (Long interspersed elements) Usually 1,000 to 10,000 bp long Occur in 20,000 to 1,000,000 copies per genome Almost 17% of the human genome SINEs (Short interspersed elements) Less than 500 bp in length Example: Alu sequence present in about 1,000,000 copies in human genome (10% of the genome) BIOLOGICAL SIGNIFICANCE OF TRANSPOSONS 1 The biological significance of transposons in evolution remains a matter of debate There are two schools of thought 1. TEs exist because they simply can! They can proliferate within the host as long as they do not harm the host to the extent that they significantly disrupt survival This has been termed the selfish DNA theory 2. TEs exist because they offer some advantage Bacterial TEs carry antibiotic-resistance genes BIOLOGICAL SIGNIFICANCE OF TRANSPOSONS 2 TEs may cause greater genetic variability through recombination TEs may cause the insertion of exons into the coding sequences of protein-encoding genes This phenomenon, called exon shuffling, may lead to the evolution of genes with more diverse functions BIOLOGICAL SIGNIFICANCE OF TRANSPOSONS 3 Transposable elements can rapidly enter the genome of an organism and proliferate quickly Example: Drosophila melanogaster A TE known as the P element was probably introduced into the species in the 1950s Remarkably, in the last 50 years, the P element has expanded throughout D. melanogaster populations worldwide The only strains without the P element are lab stocks collected prior to 1950 HYBRID DYSGENESIS Crossing D. melanogaster M strain females X P strain males Produces offspring with a variety of abnormalities (including high rate of mutation and chromosome breakage) This deleterious outcome, called hybrid dysgenesis, occurs because the P elements can transpose freely when they first enter the egg The egg does not contain any regulatory factors to prevent transposition and the sperm does not contribute cytoplasm BIOLOGICAL SIGNIFICANCE OF TRANSPOSONS 4 Transposable elements have a variety of effects on chromosome structure and gene expression Since many of these outcomes are likely to be harmful, transposition is usually highly-regulated It occurs only in a few individuals under certain conditions Agents such as radiation, chemical mutagens and hormones stimulate the movement of TEs TABLE 10.3 POSSIBLE CONSEQUENCES OF TRANSPOSITION 1 Consequence Cause Chromosome Structure Chromosome breakage Excision of a TE. Chromosomal rearrangements Homologous recombination between TEs located at different positions in the genome. Gene Expression Mutation Incorrect excision of TEs. Gene inactivation Insertion of a TE into a gene. Alteration in gene regulation Transposition of a gene next to regulatory sequences or the transposition of regulatory sequences next to a gene. Alteration in the exon content of a gene Insertion of exons into the coding sequence of a gene via TEs. This phenomenon is called exon shuffling.