Plopper Cell Biology Chapter 2
Plopper Cell Biology Chapter 2 BIOLOGY 202
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Chapter 2 Nucleic Acids 2.1 – The Big Picture Nucleotides building blocks for cells contain derivatives of the monosaccharide ribose (ribo/deoxyribo; RNA/DNA) polymers of nucleotides = nucleic acids DNA = deoxyribonucleic acid = serves as genetic material in all cells 4 major concepts in this chapter: DNA is storehouse of cell information. Relatively simple linear code enormous number of different products DNA molecule structure is relatively simple – can be learned by memorization DNA organization in a cell is relatively complex – can be organized from poorly organized to highly organized Cells have developed strategies for protecting structure and organization of DNA against damage if something is valuable, it is worth protecting. 2.2 – All of the Information Necessary for Cells to Respond to their External Environment Is Stored as DNA To be alive, cells must be able to self-replicate and self-correct Cells have to be able to sense their surroundings so they know when it is safe to replicate and that they are aware of their own functional state and know when they are damaged. To “know” these things, cells must contain information. Cellular Information cells capture and store information about their surroundings and their own physical state and is stored in the form of molecules. o cells use different types of molecules to store different types of information they need to stay alive. (i.e. DNA) DNA Used to store information for constructing other complex biological molecules such as proteins and RNA (ribonucleic acid) responsible for constructing nearly every molecule found in cells Stores most fundamental information necessary for life for every cell and organism A Cell’s DNA Is Inherited DNA stores information in a linear sequence of repeating units uses a very simple language, composed of only four molecules, commonly known as A (Adenine), C (Cytosine), T (Thymine), and G (Guanine). o Each of these letters is a molecule known as a deoxyribonucleotide, which are attached end-to- end to form very large structures in cells. When cells replicate: they divide into two parts and each of the daughter cells inherits its own complete copy of the parental cell’s DNA requires parental cell to replicate its DNA prior to cell division. In multicellular organisms such as humans, most cells (called somatic cells) only pass their DNA on to the cells that replace them during that organism’s lifetime a specialized set of cells, often called germ cells (e.g., eggs and sperm), is usually responsible for passing DNA from one organism to its offspring. Mutations in DNA Are Passed from Generation to Generation When cells replicate their DNA, they frequently make mistakes Some of these mistakes result in changes to DNA sequence. This is understandable, considering how many DNA replication events take place in an organism’s lifetime. For example, take a look at the human body: o Each human cell contains approximately 12 billion (12 × 10 ) deoxyribonucleotides in its DNA. 13 The number of cells in an average human body at any given time is estimated to be 3 × 10 , which replicate to form approximately 10 cells over the course of a person’s lifetime. Generating 10 cells from a single fertilized egg therefore requires (12 × 10 ) × 10 = 12 × 10 25 nucleotides to be replicated in the correct order. Nothing in nature can achieve level of accuracy necessary to replicate all of these nucleotides perfectly In fact, DNA replication errors are actually quite common: o in humans, the rate of inserting wrong deoxyribonucleotide in DNA sequence (point mutation) is approximately 1 per every 2 × 10 deoxyribonucleotides, or about 6 errors each time a cell replicates. Other changes in original DNA template sequence can also occur: extra deoxyribonucleotides can be inserted some can be left out large pieces of DNA can be accidentally deleted, added, and/or moved to another location in the sequence. The net effect of these changes: every cell, and everyorganism, is at least slightlydifferent from its ancestors, siblings, and other relatives. This heterogeneity in DNA sequences contributes a great deal to variation found in populations of organisms that undergo evolution by natural selection. DNA Must Be Read to Be Useful DNA is not useful in isolation it has to be READ only the portions that are “read” are meaningful they are “read” by proteins that bind to specific DNA sequences the binding changes behavior of the protein and that info. is then converted to RNA (transcribed) those RNA sequences are then translated into amino acid sequences that build to make protein For many years, scientists thought large percentage of DNA in most eukaryotic cells was useless they could find no evidence proteins would bind to these regions. More recently, scientists discovered that this so-called “junk” DNA contains characteristic patterns of repeating DNA sequences that either bind to proteins directly or control shape of neighboring DNA sequences that bind proteins much of this DNA contains deoxyribonucleotides that are chemically altered (e.g., by methylation). These chem modifications can have profound impact on which portions of DNA are read by cell, and are so important that a new field of biology has emerged for studying how these modified sequences impact phenotypes and behaviors of organisms. epigenetics DNA Information Is Packaged into Units Called Genes Most familiar form of DNA is a segment of deoxyribo-nucleotides known as a gene Gene there is no universally accepted definition for this term In this book, gene = linear sequence of deoxyribonucleotides necessary for converting portion of that sequence into complementary sequence of ribonucleotides (transcription) inside cell. Less formally a gene is a portion of DNA that can be converted into RNA, plus some additional sequences that are absolutely necessary for this conversion to happen. A gene is always a single linear sequence of deoxyribonucleotides on a single piece of DNA (cannot be fragmented into different portions of DNA scattered throughout different DNA molecules) The average length of a human gene is 10,000–15,000 nucleotides (base pairs, or bp), though there is considerable variation in size. are the best-known units of biological inheritance Gregor Mendel first discovered principles of genetic inheritance in mid-19th century was studying how individual genes of pea plants in his garden were passed from generation to generation. Heinrich Wilhelm Gottfried Waldeyer-Hartz discovered linear strands in nucleus that changed color when he added stain to cells, and he called these structures chromosomes (derived from Greek, meaning colored bodies). Thomas Hunt Morgan determined that genes are arranged on chromosomes. A single chromosome may contain thousands of genes lined up one after another, each with its own distinct packet of info. The modern definition of a chromosome = genetic element containing genes essential to cell fxn. Genetics, the field of study devoted to uncovering mechanisms governing inheritance and expression of genes o has contributed greatly to our understanding of cell behavior. Genes Are Transcribed into RNAs Genes share one important trait: some portion of their deoxyribonucleotide (DNA) sequence can be converted by a cell into a complementary ribonucleotide (RNA) sequence thru transcription results in an RNA molecule Coding sequence = portion of gene that is replicated as RNA In eukaryotes o coding sequence is often broken up into segments (exons) separated by o noncoding sequences called introns. o The process of synthesizing an RNA molecule is called transcription Many different types of RNA molecules can be encoded by genes: o Ribosomal RNA (or rRNA) molecules are an essential component of the large and small subunits of ribosomes, the molecular complexes that make proteins. In humans, the small subunit rRNA is about 1,900 nucleotides (sometimes referred to as simply bases) long, and the large subunit rRNA is about 5,000 nucleotides long. Messenger RNA (or mRNA) molecules unlike rRNAs, do not play an active role in any cellular activity they serve as templates for assembly of the ribosomes that will build a specific new polypeptide. are short-lived, intermediate copies of DNA information that are translated into proteins. Human mRNAs average 2,500 nucleotides in length. Transfer RNA (or tRNA) molecules are bridge molecules that link AAs to a specific three-nucleotide sequence on mRNA specifically deliver correct amino acids to ribosomes, where they are added to polypeptides being synthesized. Compared to rRNAs and mRNAs they interact with, tRNAs are comparatively tiny (typically about 73– 93 nucleotides long). small nuclear RNAs (snRNAs) (100–200 nucleotides long) short interfering RNAs (siRNAs) (20–25 nucleotides in length) microRNAs (miRNAs) (19–30 nucleotides long) have a wide range of functions are called noncoding RNAs because they are transcribed, but not converted into proteins o some play a role in controlling gene transcription and chromosome condensation during mitosis o others help determine location of mRNA in the cytosol. Messenger RNAs Are Translated into Proteins The combined product of rRNAs, tRNAs, and mRNAs working together newly synthesized polypeptide three-nucleotide codons in the coding sequence of mRNA are matched with anticodons in tRNA by ribosomes (including rRNA) to determine sequence of amino acids in resulting polypeptide. Despite the fact that 64 different codons are possible (4 different nucleotides can fill each position, therefore 4 or 4 × 4 × 4 = 64), each does not code for a unique amino acid. 3 codons (UAG, UAA, UGA) are designated as “stop” codons, which halt translation, and in the remaining 61 codons, redundancies ensure that only 20 different tRNAs (and amino acids) are specified by an mRNA. The average size of a human gene coding sequence is about 500–600 codons, yielding a polypeptide of 500–600 amino acids in length. Therefore, an average-sized human gene can theoretically encode at least 20500different polypeptide sequences (20 different amino acids per each of the 500 codons). In reality, the actual number of polypetides produced by any single organism is far less than this, for a good reason: most of these polypeptides would not be useful to cells. Polypeptides (either as individuals or as assembled groups) form proteins, some of the most common molecular structures in cells, and proteins have some strict requirements for how their polypeptides must behave. The information stored in DNA can undergo two transformations to become useful to cells. Transcription simply changes DNA informational sequence into a complementary RNA informational sequence. This has a profound benefit for cells: o RNA molecules are quite small (unlike DNA) and can be transported to different regions in a cell relatively easily. o multiple RNA molecules can be generated from the same DNA sequence, so a cell can configure its RNA profile by simply changing number of RNA molecules it copies from each of its genes. This is one easy way for cells to become specialized. Translation occurs when mRNA information is used to build polypeptides. It is the process of converting the ribonucleotide sequence of mRNA into an amino acid sequence in a polypeptide mRNA is the only known type of RNA that is translated. This, too, offers great benefits to cells. o DNA and RNA molecules are each composed of only 4 different subunits (nucleotides), so that the number of variations available is relatively small. o By comparison, proteins are composed of 20 different subunits (amino acids), which permit them to form much greater numbers of different molecules. allows the information in DNA to be expressed in a myriad of different proteins. Mutations in DNA Give Rise to Variation in Proteins, Which Are Acted Upon by Natural Selection Mutations have the potential to alter structure and function of RNAs and/or proteins. If occurs in the coding sequence of a gene, this change in DNA sequence is reflected by corresponding change in RNA sequence that arises from transcription. In some cases, point mutations have little or no effect on structure and function of the RNA In other cases, effects of mutations can be profound, resulting in formation of a dramatically different RNA or no RNA at all. All three types of RNAs play a role in synthesizing proteins mutations in their sequences have potential to alter sequence of amino acids created from an mRNA especially true when a sequence in coding region of an mRNA is altered this region of mRNA determines order of tRNAs it binds to thus amino acids in the new polypeptide. If coding region of mRNA is changed, the order of tRNAs that bind to it will be changed accordingly, and resulting polypeptide will have a different sequence of amino acids Example: Point mutation in hemoglobin gene that causes sickle-cell disease. o In this case, single deoxyribonucleotide change in DNA coding sequence of hemoglobin gene (the 17th nucleotide is changed from A to T) causes change in mRNA and single amino acid change in hemoglobin protein (the 6th amino acid is changed from glutamic acid to valine). o This tiny change causes RBCs to adopt “sickle” shape when concentration of oxygen in blood drops, because the HgB protein in RBCs changes from normal tetrameric configuration to long polymer strands that distort membrane of RBCs. o Sickle-shaped cells can get stuck in capillaries and/or hemolyzed (ripped open) interfering with proper circulation and causing a great deal of pain Mutations in tRNA or rRNA mitochondrial genes can also have significant effects in cells (mitochondria and chloroplasts both have their own DNA, and therefore perform transcription and translation of genes). Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) o Several mutations have been found in gene encoding tRNA that carries amino acid leucine to ribosomes in mitochondria, causing a wide range of problems (such as diabetes, degeneration of muscle fibers and nerves, and stroke-like episodes). Aminoglycoside-induced hearing loss o a single point mutation—the substitution of G for A at position 1,555—in the coding sequence of the gene for the rRNA found in small ribosomal subunit (often called the 12S subunit) is associated with a form of deafness. o The molecular mechanisms linking these mutations to their associated physiological problems are still not clear. Many different possible combinations of mutations in an organism Some can be fatal those that cause healthy cells to become cancer cells Most mutations do not cause serious problems for living cells and organisms. o The reason for this is fairly simple: typically, alterations in a DNA sequence either: (1) have little to no effect on the structure and function of the RNAs and/or proteins produced by a cell (2) have such a drastic impact on the cell that it (and possibly the entire multicellular organism in which it lives) quickly dies. o Because of this, most cells in a multicell organism, or in a population of single-celled organisms, are subtly different from previous generation of cells that divided to form them. o Most scientists believe a slow, steady rate of mutation persists for several rounds of cell division until enough mutations accumulate to generate a noticeably different cell type, and possibly even a new type of organism, if these mutations are passed on from one generation to next. In short term, this can have dire consequences for an organism: most cancers arise from cells that have acquired multiple mutations from their ancestral cells over the course of an individual’s lifetime In long term, this can have a positive impact. Each generation of cells and organisms is subtly different from its ancestors, and it is this variation that permits populations of organisms to adapt to changing environmental conditions. Evolution by natural selection, the term coined by Charles Darwin to describe the impact of the environment on the survival of individuals, only functions if a population of organisms contains some inheritable variation. The persistent mutation rate resulting from errors in DNA replication can therefore be viewed as an important tool to help ensure survival of a species. In effect, every member of a population of organisms, including humans, can be viewed as an experiment in natural selection. We all speak the same genetic language, but we are all mutants, in one way or another. 2.3 – DNA is Carefully Packaged Inside Cells Because DNA is heritable it reflects tremendous amount of information that has been gathered over the course of billions of years of evolution by natural selection. Even simple cells have hundreds of thousands of nucleotides in their DNA. To be useful as a template – nucleotides need to be accessible on demand. So this is a challenge for cells: condense DNA into a manageable size AND still permit access to each nucleotide the solution to this is complex. In this section, we will focus on the structure of the DNA double helix = the fundamental unit of DNA structural organization. DNA Is a Linear Polymer of Deoxyribonucleotides Deoxyribonucleotide = the simplest building block in DNA which makes up the double-stranded DNA molecule Start with Ribose Step 1: Replace –OH group with –H on the 2’ Carbon to form deoxyribose Step 2: Attach a base to the deoxyribose at the 1’ Carbon (that base can be Purines: Adenine or Guanine or Pyrimidines: Cytosine and Thymine) --- All 4 bases contain a N atom bonded to an H atom that is pointed downward in diagram Join the base to the rest of the structure by creating a covalent bond between the 1’ Carbon on the ribose molecule and the N atom on the base. This reaction is called dehydration (or condensation) because it also yields a single water molecule as one of its products, resulting in a deoxyribose sugar attached to a nitrogenous base and this structure is called a deoxyribonucleoside 4 different bases => 4 different deoxynucleosides Deoxyadenosine and Deoxyguanosine (Adenine and Guanine) Deoxycytidine and Deoxythymidine (Cytosine and Thymine) Step 3: Attach triphosphate to the deoxyribose at the 5’ Carbon. Create a covalent bond between 5’ Carbon and the nearest phosphate by performing another dehydration reaction resulting in a deoxynucleoTIDE. There are 2 things to remember about deoxy(ribo)nucleotides Deoxynucleotides have 1, 2, or 3 phosphate groups attached to the 5’ carbon and are named accordingly – i.e. deoxyadenosine monophosphate/diphosphate/triphosphate (dAMP, dADP, or dATP). The bond linking the phosphate to the 5’ carbon is called a phosphoester bond. Simplify polymers of nucleotides using A, G, C, T Ribonucleic acids (RNAs) are composed of subunits that closely resemble deoxynucleotides in DNA. Difference between DNA and RNA: a) Uracil is used in place of Thymine and b) nucleotides in RNA use ribose instead of DEOXYribose (AMP, ADP, ATP) ATP and GTP serve as building blocks for RNA but also serve as sources of metabolic energy and help control function of proteins A Single Strand of DNA Is Held Together by Phosphodiester Bonds Deoxynucleotides can be joined together in a linear fashion, via ester bonds.by performing dehydration reactions between phosphate group of one and the hydroxyl group on the 3’ carbon of another result is a string of deoxynucleotides linked together by an alternating sequence of phosphates and deoxyribose sugars (sugar-phosphate backbone) held together by phosphodiester bonds with nitrogenous bases. No matter how long string is – one end always has unbound 5’ carbon and other end has unbound 3’ carbon. These are called the 5’ and 3’ ends of DNA. Strand of DNA is called Deoxyribonucleic acid --- any linear sequence of the 4 deoxynucleotides arranged in a 5’ to 3’ manner. Nucleic acids can refer to DNA or RNA. DNA Forms a Double-Stranded Helix Single strands of DNA are not stable in cells. Cells stabilize DNA by organizing it. Level 1 – Simplest form of stable DNA in cells double stranded DNA molecule where the 2 strands run antiparallel to eachother (one strand of 5’ to 3’ is alongside one strand that runs 3’ to 5’) and are held together by hydrogen bonds between O and N atoms in the complementary bases to form base pairs. The absence of the –OH group on the 2’ carbon of deoxyribose allows the 2 DNA strands to twist around one another to form a helix. Complementary base pairing holds the 2 strands of DNA together. Two strands in the double helix are stabilized by the hydrogen bonds between complementary bases. Double helix contains 2 different grooves. Wider groove is called the major groove, the narrower groove is called the minor groove. These grooves are important because they form attachment sites for DNA binding proteins. DNA binding proteins often contain finger-like structures that fit into these grooves allow DNA binding proteins to slide back and forth in the grooves as they search for specific sequence of deoxynucleotides they are targeting. Twisting of the DNA strands results in a periodicity of approximately 3.4 nm (or 34 Å) means that there are approximately 10.5 base pairs per turn of the helix. This matters because many DNA binding proteins bind to short DNA sequences (6 base pairs or fewer). Because these sequences are shorter than a single turn of the helix, they seem more-or-less linear to DNA binding proteins, and this makes them easy to detect. DNA is composed of a large number of atoms changes in even a few atoms result in noticeable variations in DNA structure. a region of DNA encoded by several A–T base pairs will have slightly different shape than one encoded by G–C base pairs, even though both pairs are perfectly aligned. each base pair imparts its own shape to overall molecule two segments of DNA made up of different deoxynucleotide sequences also have slightly different shapes which allows proteins to “know” which regions of DNA to bind to. every different sequence of deoxyribonucleotides has its own unique shape. proteins that bind to specific sequences of DNA can therefore slide along a strand of DNA until they find exact shape that fits their binding site. Even very minor changes in atomic structure of deoxynucleotides can have profound effects on overall DNA shape o one common cause of DNA mutation is the loss of a single amino group (–NH) from 2he base of a single deoxynucleotide. DNA Can Be Supercoiled to Form at Least Three Different Structures Discovering the double-stranded, helical organization of DNA was one of the most significant advances in biology of the 20 century. A considerable amount of the data used to deduce this structure came from crystals of DNA grown in a lab. These crystals demonstrated that dsDNA can form at least 3 different types of double helix: B-DNA: crystallized under condition of high relative humidity (92%) A-DNA: crystallized under condition of low relative humidity Z-DNA All 3 types have major grooves and minor grooves Because interior of cell is entirely saturated with water, it is likely most of the DNA in a living cell adopts a shape very similar to B-DNA. Small regions of Z-DNA have been detected in cells still unclear what significance this conformation plays in chromosome function. A-DNA and B-DNA are right-handed Z-DNA is left-handed suggested that Z-DNA regions may serve to reduce amount of effort required to unwind B-DNA in areas of the chromosome that are frequently copied during transcription. 2.4 – DNA Packaging Is Hierarchical DNA packaging Problem – how to store lots of information, accumulated over billions of years of evolution, in form of linear sequences of deoxyribonucleotides (very, very long) roughly 12 billion deoxyribonucleotides in an average human cell form about 6 billion base pairs How can all that info be packed into a single cell without getting all jumbled up? We will describe Levels 2 through 5 of DNA organization here. DNA Is Bound to a Protein/RNA Scaffold First part of strategy for packaging DNA in cells support it with elaborate infrastructure made of protein and RNA to keep DNA from getting hopelessly tangled and thus useless. Chromatin = complex formed by proteins (50% of mass), RNA, and associated DNA in the nuclei of eukaryotes in the nucleoid in mitochondria, chloroplasts of prokaryotes and of Archaea Double-Stranded DNA Is Wrapped Around Histone Proteins to Form a Small Particle The best-known set of structural proteins belongs to the histone family. Histones are found in all organisms are thought to be some of earliest proteins to appear during evolution. when associated with DNA form spools, similar to those used to store thread, string, wire, etc. (Level 2 of DNA organization) o In eukaryotic cells, these spools are composed of two copies each of four different histones (named H2A, H2B, H3, and H4). contain many positively charged amino acids, which attract them to the negatively charged backbone of DNA that is largely sequence independent. o dsDNA molecule is wrapped around a histone “spool” approximately 1.7 times, to form a core particle o An additional histone, either H1 or H5, “pins” DNA to the core particle, resulting in a structure called a nucleosome. In eukaryotes, nucleosomes contain around 167 base pairs of DNA separated by short stretches (20–50 base pairs) of DNA called linker DNA string of beads appearance. Similar structures are found in prokaryotic cells, where DNA is wrapped around a different set of histone protein spools. Wrapping DNA in this fashion causes DNA double-helical strand to become shorter and thicker o the length is reduced approximately 7-fold o width increases from 2 nm to about 11 nm. o These “beads-on-a-string” structures also contain proteins in addition to histones in both prokaryotes and eukaryotes. Nucleosome Structure Can Be Modified by Cells Does wrapping DNA around a spool have any negative consequences? Remember, the goal is to compact DNA without compromising a cell’s ability to access the genetic info. Because so much of DNA comes into contact w/spool, most of it is inaccessible to other DNA-binding proteins, thereby negating beneficial effects of these spools. Cells use three different mechanisms to address this problem: First in eukaryotes, DNA can be partially unwrapped from nucleosome by members of the SWI/SNF family of proteins. o use ATP energy to move core particle a short distance along DNA freeing up any base pair sequences that may have been buried in core particle o At least two other families of proteins participate in chromatin remodeling Second Histone remodeling is very important way to modify shape of the nucleosome, and is based on chemically modifying histone proteins in nucleosome. o A variety of proteins are capable of attaching relatively small molecules (methyl groups, acetyl groups, or phosphate groups) to tails of histones, which causes them to change their shape o In some cases, modifications make it easier to access core particle DNA, and in other cases, the opposite is true. Third methyl groups may also be added directly to bases A (prokaryotes) or C/G (eukaryotes) in DNA, a process called DNA methylation. These three mechanisms modifystructure and 3-D shape of chromatin w/o altering sequence of deoxynucleotides topic in field of epigenetics, because it is believed patterns of these modifications help control the expression of genes. When we break cells open and spread out contents See beads-on-a-string structure under the microscope In intact cells: nucleosomes are clustered together in highly ordered fashion to form series of similar configurations, all of which are called the 30-nm fiber. (In prokaryotes, similar 40-nm fibers form from clusters of the “beads” in the nucleoid.) These fibers represent Level 3 of DNA organization. The reason that multiple configurations have the same name is that scientists are still not entirely sure how many of the configurations are present in living cells (the electron microscope, which is a common tool for observing chromatin, cannot be used to view living cells). These fibers will form spontaneously in a test tube if the salt concentration of the buffer is kept low enough, demonstrating that no additional proteins or metabolic energy are required. Level 3 of DNA organization (The 30-nm fiber) is held together by electrostatic interactions between different histones. o Example, a negatively charged region on histone H4 binds to positive region in a histone H2A/H2B complex in another nucleosome, drawing two nucleosomes together and further compacting chromosome results in additional shortening and thickening of the chromosome, and increases packing density to about 42 times that of double-stranded DNA alone. Level 4 of DNA organization 30-nm fibers are attached to a protein-RNA scaffold (also called a matrix) at intervals of 10-30 μm that keeps them organized Forming so-called loop domains of approximately 60 kilobases in length (are approximately 750-fold more compact than B-DNA) o how these loops are attached to the scaffold is not known, but attachment occurs at DNA sequences called MARs (matrix attachment regions) or SARs (scaffold attachment regions). These sequences typically contain large number of A–T base pairs, but are otherwise not very similar. A number of proteins have been isolated from chromosome scaffold structure is sensitive to enzymes that digest an unknown form of RNA, but it is not yet clear how these molecules assemble to form mature structure. Chromatin Is Packaged into Highly Condensed Chromosomes Eukaryotic cells adopt an additional means for organizing DNA that most prokaryotes do not use they cut their DNA up into several chromosomes. Human beings are considered to be diploid o meaning they contain two copies of each chromosome o these are organized as 2 copies each of 22 autosomes and 1 pair of sex chromosomes. chromosomes are very large bundles of DNA with distinctive shapes that change over the course of a cell’s life During mitosis, these chromosomes condense to form their familiar X-shaped structures, and they decondense once mitosis is complete This condensation/decondensation is analogous to packing one’s belongings into a small, compact space (e.g., a suitcase for a trip), then unpacking once the journey is complete. These observations reveal two important things DNA organization is dynamic: chromosomes can be tightly bundled or loosely bundled as necessary during a cell’s lifetime Implication is that there must be some machinery responsible for controlling this bundling. Consider how important this machinery is during mitosis, a eukaryotic chromosome can be condensed into a structure that is about 15,000 to 20,000 times shorter than its unwound length. o Changes in the length of a chromosome result from complex series of folding and twisting events designed to prevent individual strands from becoming tangled. Heterochromatin Is a Form of Tightly Packed DNA in Eukaryotic Cells We have been describing only first few levels of DNA folding: wrapping around a spool twisting of the spooled DNA into 30- to 40-nm fibers further folding to yield loop domains. (prokaryotic chromosome is thought to consist entirely of these structures) Levels 1–4 of DNA organization are called euchromatin in eukaryotes they can be easily accessed by proteins responsible for replicating the chromosomes in preparation for cell division, or by proteins responsible for reading a strand of DNA to make RNA (i.e., transcription). DNA sequences organized as a form of euchromatin are easy to use o helps explain why prokaryotic cells can alter their gene expression patterns fairly rapidly, compared to most eukaryotes their entire DNA is already easily accessible. Eukaryotes go even further in compacting their chromosomes. unlike in prokaryotes, a considerable amount of DNA in eukaryotic chromosomes is actually rather useless to a cell o may contain genes or fractions of genes that were important for our distant ancestors, but are no longer useful. o may contain genes that are only useful during early embryonic development or in specialized cells (e.g., humans form gill-like structures only during early development, usually only nerve and muscle cells make neurotransmitter receptors while only bone cells make bone proteins). o Once a cell commits to a specific developmental fate (e.g., nerve, muscle, or bone), it no longer needs access to at least some genes required for other fates (e.g., skin or liver) o A nerve cell can afford to “pack up” the portions of its DNA containing instructions necessary for functioning as a liver cell, because it has no expectation of ever using them. Level 5 of DNA organization Advancing to the next stage of DNA condensation, Level 5, is a very big step for these cells. Any portion of a chromosome that condenses past point of loop domains becomes essentially inactive Twisting DNA into Heterochromatin Requires Metabolic Energy Turning into Heterochromatin additional condensation of DNA is accomplished by twisting loop domains into shorter and thicker filaments. Several different structures are possible, depending on extent of twisting used. The degree of this compaction has been estimated to be between 250- and 10,000-fold. o We will break this range into two parts: 1. those areas of condensed DNA found in cells that are not actively dividing (also called interphase) are Level 5A 2. more compact chromosomes are required for cells to undergo mitotic or meiotic phase of cell division, and this extra degree of compaction is Level 5B. All of these Level 5 structures are called heterochromatin o differentiated from euchromatin because they appear as blobs of varying darkness in an electron microscope o Unlike condensation of nucleosomes to form 30-nm fibers, this condensation requires additional proteins two good examples are those belonging to the SMC (structural maintenance of chromosomes) family of proteins. Condensins = responsible for general chromosome structure, along with chromosome condensation at mitosis. Cohesins = plays an important role in condensation of yeast chromosomes during cell division and regulates access to genes in virtually all eukaryotes. Some of these proteins require energy to function, so formation of heterochromatin is metabolically costly. In both groups, ATP hydrolysis is thought to supply energy necessary for them to assist in condensation, though it is still not clear exactly what role it plays. Scientists discovered another ATP-dependent group of proteins that play crucial role in chromosome condensation prior to mitosis These proteins are responsible for reading highly repetitive sequences of DNA to synthesize special forms of double-stranded RNA called short interfering RNA (siRNA) until then most researchers believed siRNAs only controlled gene transcription While the exact mechanism has yet to be determined, the siRNA-forming proteins recruit and bind to still another group of proteins that modify histones an essential step in heterochromatin formation. DNA Is “Silenced” in Heterochromatin Though exact mechanism of heterochromatin formation is not known, the best-known method is “silencing” DNA in a nucleosome. So far, at least two different mechanisms for silencing DNA have been identified: First mechanism: o an enzyme called histone deacetylase removes an acetyl group (found in many actively transcribing genes) from histone H3. o A protein called histone methyltransferase attaches a methyl group to the ninth amino acid (a lysine, abbreviated K) on histone H3 (this is sometimes abbreviated as H3K9me). o Finally, a third protein (in mammals it is called heterochromatin protein 1, or HP1) attaches to the newly methylated histone H3. When this histone is deactylated and methylated, its shape changes. This triggers a conformational change in the entire core particle, such that the DNA attached to the core particle can no longer be transcribed; it is now silenced. As additional nucleosomes next to the newly silenced section undergo the same modifications, the silencing can extend to larger stretches of DNA. Second mechanism: o a protein called Rap1 binds to a portion of DNA. o resulting change in Rap1’s shape allows two additional proteins named Sir3 and Sir4 to attach to it, and they in turn bind to histones H3 and H4. o This creates a change in the shape of the core particle and facilitates the binding of additional Sir3/Sir4 complexes to adjacent nucleosomes. Note that in both strategies, changing the structure of the nucleosome core particle by altering the configuration of histones is a key step. Some Regions of Eukaryotic Chromosomes Are Always Silenced In eukaryotes portions of chromosomes are never active called constitutive heterochromatin (in biology, constitutive means “constantly produced”). A minimal amount of transcription, such as for siRNAs, takes place in these regions, but none of the resulting RNA transcripts are ever translated these regions play important roles in maintaining structure and organization of a chromosome during mitosis. Chromosome the centromere region of the chromosome is essential for proper attachment of the chromosome to the microtubule spindle during mitosis telomere regions protect ends of the chromosome from damage Both of these regions bind to a number of different proteins, but only unwind completely during DNA replication. 2.5 - The Nucleus Carefully Protects a Eukaryotic Cell’s DNA DNA is found in own organelle in eukaryotes Nucleus Nucleus must simultaneously allow access to a large number of proteins responsible for reading, replicating, repairing, and remodeling the DNA (e.g., histones) while also protecting the DNA from a host of environmental dangers. How might a cell do this? The Nuclear Pore Complex Restricts Access to the Interior of the Nucleus Nuclear pore complex, or NPC, penetrates both membranes surrounding the nucleus. o Each nucleus contains hundreds to thousands of NPCs, the primary function of which is to control the molecular traffic of objects >30 kilodaltons in size between the nucleus and the cytoplasm. o Several different types of molecules pass through these pores, including large structures such as RNA molecules (synthesized in the nucleus and exported) and proteins (recycled between the cytosol and nucleoplasm), as well as the large and small ribosomal subunits (synthesized in nucleoli, regions in the nucleoplasm specialized for this purposeFigure 2-25, and even viruses. o The key to passing through the NPC is a specific signal that is attached to these molecules; without the signal, the molecules are blocked by the complex. o Transport of these molecules requires the expenditure of metabolic energy; the mechanisms governing how these molecules are marked and transported through NPCs are quite complicated o Molecules smaller than 30 kilodaltons, such as nucleotides, sugars, ions, and water, can diffuse freely through the NPC. This strategy protects DNA because the kinds of molecules most likely to damage it (e.g., proteins that degrade nucleic acids) are restricted from entering the nucleus unless they are needed. Nuclear Lamin Proteins Form a Protective Cage around the Chromosomes Network formed by proteins called nuclear lamins are members of the intermediate filament family of proteins are characterized by their tremendous resistance to mechanical force when nuclear lamins are mutated and no longer functional, this has dramatic consequences for cells, especially muscle cells that are subject to great mechanical force. are found in most multicellular eukaryotes (except plants) and in some unicellular eukaryotes (like yeast) lamin proteins bind to NPCs inside the nucleus and to chromatin On EM, nuclear lamins give inner nuclear membrane a rough, fibrous appearance. complexes are composed of three different lamin proteins, called lamins A, B, and C. Mounting evidence suggests lamin proteins interact directly with both chromatin and even the DNA One of the chromatin proteins is HP1 which binds lamin B. Nuclear matrix The lamin-chromatin complex is part of a network of proteins called the nuclear matrix, which coats chromosomes plays a role in regulating gene expression lamins help organize chromosome unwinding during telophase of mitosis also. 2.6 – Chapter Summary To stay alive, cells must do 2 things: 1. respond appropriately to external signals and internal programs 2. maintain their internal environment Nearly all of the molecules responsible for 1) and 2) are RNA or proteins and instructions for creating these molecules are stored in DNA (a relatively simply polymer). Parental cell DNA Each time a cell divides, daughter cells inherit a copy, which is slightly modified in successive generation of cells by mistakes (mutations) made during replication process. instructions in DNA are organized into units called genes cells read these genes to produce several types of RNAs (transcription) proteins (translation of mRNA). o Mutated genes altered RNAs and proteins when they are transcribed and translated these differences in RNAs and proteins yield variation in cellular phenotypes that act upon natural selection in each generation of cells and organisms. DNA heritable material acted upon by evolution encodes instructions for producing all of RNAs and proteins a cell will require during its lifetime (may also encode additional unused genes) it is typically an enormous molecule relative to cell that harbors it. Complete DNA molecule = chromosome is made up of combinations of four subunits, called deoxyribonucleotides, which form two antiparallel strands held together by hydrogen bonds. One of these two strands contains coding sequence of a gene. To ensure genes can be easily accessed while also compacting them enough to fit into a cell, DNA is supported by elaborate protein/RNA scaffold, called nucleoid in prokaryotes and chromatin in eukaryotes. o Histone proteins are at heart of these scaffolds, and DNA wraps around “spools” of histones. Chemical modifications of histones and DNA bases play important role in controlling which sections of DNA molecules are read by transcription machinery. o In some eukaryotes, portions of chromosomes are condensed and modified so they don’t undergo transcription = heterochromatin (not found in prokarys) o Euchromatin = transcriptionally accessible regions. Eukaryotic cells: store DNA in nucleus and restrict access to it with nuclear pore complexes = highly selective structures that control traffic of proteins, nucleic acids, and other large molecules into and out of nucleus. Some eukaryotes also contain lamins, which are proteins in their nucleus, as added form of support and protection for DNA. o Lamin proteins are strong proteins that resist mechanical forces imposed on nucleus o recent evidence suggests they help control gene expression by binding chromatin and specific DNA sequences.
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