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BIO214 Chapter 5 Objectives After completing Chapter 5, you should be able to answer the following questions on an exam: 1. How did Griffith’s and Avery and MacLeod’s experiments demonstrate that DNA was the transforming principle? § Griffith’s experiment- hereditary information can be transferred from one bacterium to another § He was studying Streptococcus pneumonia, a bacterium that causes pneumonia. § When grown in laboratory, pneumococci come in two forms: a pathogenic form that causes a lethal infection when injected into animals, and a harmless form that is easily conquered by the animal’s immune system and does not produce an infection § He injected various preparations of these bacteria into mice. He injected both heat-killed pathogenic bacteria and live harmless bacteria into the same mouse, died of pneumonia, their blood was teeming with live bacteria of the pathogenic form. § The heat killed pneumococci had somehow converted the harmless bacteria into the lethal form, he could grow these transformed bacteria in culture and they remained pathogenic. § Avery and MacLeod- The molecule that transforms R to S-strain is DNA § Discovered that the harmless pneumococcus could be transformed into a pathogenic strain in a culture tube by exposing it to an extract prepared from pathogenic strain. § They successfully purify the transforming principle from this soluble extract and to demonstrate that the active ingredient was DNA. Because the transforming principle caused a heritable change in the bacteria that received it, DNA must be the stuff of which genes are made. § They showed that enzymes that destroy proteins and RNA did not affect the ability of the extract to transform bacteria, while enzymes that destroy DNA inactivated it. § Their purified preparation changed the bacteria permanently- DNA from the pathogenic species was taken up by the harmless species, and this change was faithfully passed on to subsequent generations of bacteria. 2. How is genetic information encoded in DNA? § Chromosomes- composed of protein and nucleic acids § Information is encoded in the order, or sequence, of the nucleotides along each DNA strand. 3. What type of bond connects nucleotides together in DNA and RNA? § For DNA nucleotides are connected through sugar and phosphate groups. The numbering based on numbering of sugar ring. Phosphate connected to 5’OH group of sugar. § The nucleotides are covalently linked together in a chain through the sugars and phosphates, which forms a backbone of alternating sugar- phosphate-sugar-phosphate. § Each polynucleotide chain in DNA can be thought of as a necklace: a sugar-phosphate backbone, strung with four types of beads (A, C, G, T). 4. What are three differences between the structure of DNA and RNA? § DNA is a double stranded polymer- A, C, G, and T. It has base pairing the sugar is deoxyribose 5. What are the base pairing rules for DNA? RNA? Do RNA molecules have 3-D structures? § DNA: A always pairs with T, and G always pairs with C. § In each case, a bulkier two-ring base (a purine) is paired with a single-ring base (pyrimidine). Each purine-pyrimidine pair is called a base pair, and this complementary base pairing-enables the base pairs to be packed in the energetically most favorable arrangement in the interior of the double helix § In this arrangement, each base pair has a similar width, thus holding the sugar-phosphate backbones an equal distance apart along the DNA molecule. § The members of each base pair can fit together within the double helix because the two strands of the helix run antiparallel to each other, they are oriented with opposite polarities. The antiparallel sugar-phosphate strands then twist around each other to form a double helix containing 10 base pairs per helical tuen. § Each strand of a DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand 6. How are the two strands of the DNA double helix held together? § Strands of the double helix are anti-parallel § Hydrogen bonds connect the two strands § The two polynucleotide chains in the DNA double helix are held together by hydrogen-bonding between the bases on the different strands. All of the bases are on the inside of the double helix, with the sugar-phosphate backbones on the outside. 7. Why is DNA considered a polar molecule? This polarity in a DNA strand is indicated by referring to one end as the 3’ end and the other as the 5’ end. This convention is based on the details of the chemical linkage between the nucleotide subunits. 8. How is DNA stored in eukaryotic cells? § DNA molecules are packaged into chromosomes. These DNA molecules fit inside the nucleus, but after they are replicated, they can be apportioned between the 2 daughter cells at each cell division. § The task of packaging DNA is accomplished by specialized proteins that bind to and fold the DNA, generating a series of coils and loops that provide increasingly higher levels of organization and prevent the DNA from becoming a tangled mess. § It is compacted in a way that allows it to remain accessible to all the enzymes and other proteins that replicate it, repair it, and control the expression of genes. § The DNA in the nucleus is distributed among a set of different chromosomes. Each chromosome consists of a single, long, linear DNA molecule associated with proteins that fold and pack the fine thread of DNA into a more compact structure. The complex of DNA and protein is called chromatin. § Human cells each contain two copies of each chromosome, one from mother and one from father. § Each chromosome can be painted a different color using sets of chromosome-specific DNA molecules coupled to different fluorescent dyes. This involves a technique called DNA hybridization, which takes advantage of complementary base-pairing. 9. What segments are required for a chromosome to be maintained and replicated? § Centromeres- allows one copy of each duplicated chromosome to go to each daughter cell during replication. Centromere allows duplicated chromosomes to be separated during M phase. The DNA coils up, adopting a more and more compact structure, forming highly compacted or condensed, and mitotic chromosomes. Duplicated chromosomes can be most easily visualized. § Telomeres- repeated sequences at tips, highly conserved, enzyme that adds repeats is telomerase. Has protective caps- interaction between chromosomes and nuclear envelope. Prevent chromosome fusion. o Telomeres in aging- cells in culture- don’t divide indefinitely- dramatic decrease in telomere length o Same in elderly adults- no telomerase- telomere shortening- to ‘crisis’ o Add telomerase: extend lifespan o But telomere shortening: protect from cancer- 90% of cancers: active telomerase enzyme o Telomere lengths also affected by stress- lower ranking hyenas- increased stress- have to work harder for food- results in more stress hormones, peroxides, and reactive O2 species- results in shorter telomeres- leads to cell death § Telomeres are at each of the 2 ends of a chromosome. They contain repeated nucleotide sequences that are required for the ends of chromosomes to be replicated. They also cap the ends of the DNA molecule, preventing them from being mistaken by the cell as broken DNA in need of repair. § Replication Origin- replication of the DNA begins; eukaryotic chromosomes contain many replication origins to ensure that long DNA are replicated rapidly. 10.What two types of molecules together make up chromatin? DNA and proteins 11.What is a nucleosome and how is it constructed? § Nucleosomes are the basic units of chromosome structure § Thick fiber, 30 nm, isolated from interphase nucleus § Unpacked fiber-beads on a string § Histones are responsible for the first and most fundamental level of chromosomal packing, the nucleosome. § The structure of the nucleosome core particle was determined after first isolating nucleosomes by treating chromatin in its unfolded beads on a string form with enzymes called nucleases, which break down DNA by cutting the phosphodiester bonds between nucleotides. Only the exposed DNA between the core particles- the linker DNA- is degraded, allowing the core particles to be isolated. § An isolated nucleosome core particle consists of a complex of 8 histone proteins- 2 molecules each of histones H2A, H2B, H3, and H4, and a stretch of double-stranded DNA, 147 nucleotide pairs long that winds around this histone octamer. § The formation of nucleosomes converts a DNA molecule into a chromatin thread that is approximately one-third the length of the initial piece of DNA, and it provides the first level of DNA packing. § 12.How does the amino acid composition of histones allow the proteins to interact with DNA? § Histones- basic proteins- R, K § Five classes- H1, H2A, H2B, H3, and H4 § Very conserved- only two differences in H4 § DNA and histones= nucleosomes § 146 bp DNA around histone octamer § H1= linker histone § DNA and Histones- histone octamer- § With H1, 168 bp DNA § Basic amino acids interact with DNA § 10 nm nucleosome will hold 70 nm of DNA, packaging ratio of 7:1 § An isolated nucleosome core particle consists of a complex of 8 histone proteins- 2 molecules each of histones H2A, H2B, H3, and H4, and a stretch of double-stranded DNA, 147 nucleotide pairs long that winds around this histone octamer. § The formation of nucleosomes converts a DNA molecule into a chromatin thread that is approximately one-third the length of the initial piece of DNA, and it provides the first level of DNA packing. § All four of the histones that make up the octamer are relatively small proteins, with a high proportion of positively charged amino acids. The positive charges help the histones bind tightly to the negatively charged sugar-phosphate backbone of DNA. § Each of the histones in the octamer has a long, unstructured N-terminal amino acid tail that extends out from the nucleosome core particle. 13.What is a 30-nm fiber, and how is it constructed? Chromatin fiber 14.How is the 30 nm fiber further packaged? Packaged in a zig-zag model § Short region of DNA double helix § “beads-on-a-string” form of chromatin § chromatin fiber of packed nucleosomes § chromatin fiber folded into loops § entire mitotic chromosome § net result- each DNA molecule has been packaged into a mitotic chromosome hat is 10,000-fold shorter than its fully extended length § Nucleosomes are further packed on top of one another to generate a more compact structure such as chromatin fiber. This additional packing of nucleosomes into a chromatin fiber depends on a fifth histone, called histone 1, which is thought to pull adjacent nucleosomes together into a regular repeating array. This linker histone changes the path the DNA takes as it exits the nucleosome core. § 15.What are the levels of chromosome packaging? § The chromatin fiber is folded into a series of loops, and that these loops are further condensed to produce the interphase chromosome, this compact string of loops is thought to undergo at least one more level of packing to form the mitotic chromosome. 16.What are heterochromatin and euchromatin? § Heterochromatin is the most highly condensed form of interphase chromatin. Makes up of about 10% of an interphase chromosome, it is concentrated around the centromere region and in the telomeres at the ends of the chromosomes § The rest of the interphase chromatin is called euchromatin- refers to chromatin that exists in a more decondensed state than heterochromatin. § Each type of chromatin structure is established and maintained by different sets of histone tail modifications that attract distinct sets of nonhistone proteins. Once it has been established, heterochromatin can spread because these histone tail modifications attract a set of heterochromatin-specific proteins, which then create the same histone tail modifications on adjacent nucleosomes. These modifications recruit more of the heterochromatin-specific proteins, causing a wave of condensed chromatin to propagate along the chromosome. Extended regions of heterochromatin can be established along the DNA. 17.What is X-inactivation? § Inactivated X-chromosome is a barr body, when a gene is turned off. 18.What are chromatin remodeling complexes, and how do they influence gene expression? § Chromosome remodeling complexes push DNA along nucleosomes- loosens DNA, more accessible § Turn off complexes in mitosis § Chromatin-remodeling complexes-protein machines that use the energy of ATP hydrolysis to change the position of the DNA wrapped around nucleosomes. The complexes, which attach to both the histone octamer and the DNA wrapped around it, can locally alter the arrangement of nucleosomes on the DNA, making the DNA either more accessible or less accessible to other proteins in the cell. § These modifications can serve as docking sites on the histone tails for a variety of regulatory proteins. Different patterns of modifications attract different proteins to particular stretches of chromatin. Specific combinations of tail modifications and the proteins that bind to them have different meanings for the cell: genes should be expressed or silenced. § The histone-modifying enzymes work in concert with the chromatin remodeling complexes to condense or decondense stretches of chromatin. 19.How is chromatin structure dynamic? How is it heritable? § Regions of the chromosome that contain genes that are being expressed are generally more extended, while those that contain silent genes are more condensed. The detailed structure of an interphase chromosome can differ from one cell type to the next, helping to determine which genes are expressed. BIO214 Chapter 6 Objectives 1. How does each of the two DNA strands act as a template for the synthesis of the other strand? § The strands separate, new bases can be added in § Original strands remain intact § Each new daughter DNA is one old strand and one new strand § Each strand of DNA can serve as a template for the synthesis of a new complementary strand. The two strands separate, and in turn can be a template for two new strands. This enables the cell to copy or replicate its genes by passing them on to its descendants. 2. Where does replication start? § DNA begins at replication origins- at specific sites § Bacteria and yeast- AT rich, easy to open, 100 base pairs, attract initiator proteins § Bacterial: one origin- human genome:10,000 origins § Initiator proteins attracts other components- function as a protein machine § Initiator proteins recognize sequences, pry strands apart § New DNA synthesis occurs at replication forks § Replication bubble starts at origin- two forks form in each bubble § Synthesis is bidirectional and rapid- 1000 nucleotide pairs/sec in bacteria, only 100 nucleotide pairs/sec in humans § The process of DNA synthesis is begun by initiator proteins that bind to specific DNA sequences called replication origins. The initiator proteins pry the two DNA strands apart, breaking the hydrogen bonds between the bases. They separate a short length because the individual hydrogen bonds are weak. The initiator proteins attracts a group of proteins that carry out DNA replication. These proteins form a replication machine, in which each protein carries out a specific function. 3. What is the template strand of DNA? § Each strand of DNA can serve as a template for the synthesis of a new complementary strand. The two strands separate, and in turn can be a template for two new strands. This enables the cell to copy or replicate its genes by passing them on to its descendants. 4. What does ‘semi-conservative replication’ mean? § Each of the daughter DNA double helices ends up with one of the original strands plus one strand that is completely new (semiconservative) 5. How many replication forks are formed at each origin of replication? How many polymerases are operating at each replication fork?? § Each replication bubble has 2 yorks 6. In what direction does DNA polymerase synthesize new DNA? Why? § Synthesis from 5’à 3’, nucleotides enter as dNTPs- provides energy § Newly synthesized strands are of opposite polarity- DNA remains anti- parallel § The incoming nucleotide is linked to the free 3’ hydroxyl on the growing DNA strand. § Proofreading- for a DNA polymerase to function as a self-correcting enzyme that removes its own polymerization errors as it moves along the DNA, it must proceed in the 5’ to 3’ direction. 7. What provides the energy for synthesizing DNA? § When the nucleotides enter as dNTPs 8. What are the leading and lagging strands? Why is synthesis of DNA on the lagging strand referred to as discontinuous? § Leading strand- one primer- multiple primers needed for lagging strand § All DNA polymerases add new subunits only to the 3’ end of a DNA strand. A DNA chain can only be synthesized from 5’ to 3’. But one strand is going from 5’ to 3’ and then other is 3’ to 5. The DNA strand that is incorrect is made discontinuously, in successive, separate, small pieces- with the DNA polymerase moving backward so that each new DNA fragment can be polymerized in the 5’ to 3’ direction. § The resulting small DNA pieces- Okazaki fragments- are later joined to form a continuous new strand. The DNA strand that is made discontinuously in this way is called the lagging strand because the backstitching imparts a slight delay to its synthesis; the other strand, which is synthesized continuously is called the leading strand. 9. What is an Okazaki fragment, and how does it tie in with discontinuous synthesis on the lagging strand? What role does DNA ligase play? § Two forks, moving from the center- DNA synthesis always proceeds from 5’ à 3’. One strand is synthesized discontinuously-in small pieces § Pieces are called okazaki fragments-later stitched together § Dna ligase reforms phosphodiester backbone, uses energy from ATP hydrolysis § All DNA polymerases add new subunits only to the 3’ end of a DNA strand. A DNA chain can only be synthesized from 5’ to 3’. But one strand is going from 5’ to 3’ and then other is 3’ to 5. The DNA strand that is incorrect is made discontinuously, in successive, separate, small pieces- with the DNA polymerase moving backward so that each new DNA fragment can be polymerized in the 5’ to 3’ direction. § The resulting small DNA pieces- Okazaki fragments- are later joined to form a continuous new strand. The DNA strand that is made discontinuously in this way is called the lagging strand because the backstitching imparts a slight delay to its synthesis; the other strand, which is synthesized continuously is called the leading strand. § 10.What are the functions of RNA primers, and what is the role of the enzyme ‘primase?’ § Primase synthesizes RNA primers § To begin a new DNA strand- an enzyme that begin a new polynucleotide strand simply by joining 2 nucleotides together without the need for a base- paired end. This enzyme doesn’t synthesize DNA, it synthesizes RNA using the DNA as a template. It serves as a primer for DNA synthesis and the enzyme that synthesizes the RNA primer is called primase. § Primase is an example of RNA polymerase, an enzyme that synthesizes RNA using DNA as a template. § Because U can form a base pair with A, the RNA primer is synthesized on the DNA strand by complementary base-pairing in exactly the same way as is DNA. § For the leading strand, RNA primase is needed only to start replication at a replication origin. On the lagging strand, new primers are needed to keep polymerization going because DNA synthesis is discontinuous. § DNA polymerase adds a deoxyribonucleotide to the 3’ end of each primer to start a new Okazaki fragment. § Enzymes are used to quickly remove the RNA primer, replace it with DNA, and join the fragments together. A nuclease degrades the RNA primer, a DNA polymerase called a repair polymerase then replaces this RNA with DNA and the enzyme DNA ligase joins the 5’-phosphate end of one DNA fragment to the 3’ OH end of the next. § Primers have lots of mistakes, they are to be automatically removed and replaced by DNA. § 11.On the replication fork below, be able add in the leading strands and lagging strands, and correctly indicate the direction of DNA synthesis. 12.the ‘proof-reading’ activity of DNA polymerase? What does it do during replication of DNA? § DNA polymerase can correct its mistakes- proofreading-checks before adding next nucleotide § Proofreading takes place at the same time as DNA synthesis. Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired to the template strand. If not, the polymerase clips off the mispaired nucleotide and tries again. Carried out by a nuclease that cleaves the phosphodiester backbone. 13.How are the very ends of chromosomes replicated on the lagging strands of DNA? Telomerase § When the final RNA primer on the lagging strand is removed, there is no way to replace it. Eukaryotes solve it by having long, repetitive nucleotide sequences at the ends of their chromosomes, which are incorporated into structures called telomeres. These telomeric DNA sequences attract an enzyme an enzyme called telomerase to the chromosome ends. Using an RNA template that is part of the enzyme itself, telomerase extends the ends of the replicating lagging strand by adding multiple copies of the same short DNA sequence to the template strand. This extended template allows replication of the lagging strand to be completed by conventional DNA replication. 14.Why is DNA repair necessary? § So mutations don’t happen, can ruin lives § If left unrepaired, could lead either to the substitution of one nucleotide pair for another as a result of incorrect base pairing during replication or to the deletion of one or more nucleotide pairs in the daughter DNA strand after DNA replication 15.How does mismatch repair work? How is the incorrect nucleotide identified? § Mismatch repair corrects errors made when replicating the DNA. Incorrect nucleotide will be incorporated next round, repair the new strand § First, identify the mistake- mismatch distorts backbone § Identified by mismatch repair proteins § Want to repair the newly synthesized strands- new strands in bacteria lack chemical modification, in other cells, other strategies to distinguish the newly synthesized strand 16.Describe thymine dimers. What happens if thymine dimers are not repaired? § Ultraviolet radiation in sunlight can cause the formation of thymine dimers. Two adjacent thymine bases have become covalently attached to each other to form a thymine dimer. § If not repaired-it would stall the DNA replication machinery at the site of the damage. 17.Describe what happens in depurination and what the consequences are if the mistake is not repaired. § Depurination- can result in the loss of a base pair § Depurination removes a purine base from a nucleotide, giving rise to lesions that resemble missing teeth. Removes g or a 18.Describe what happens in deamination and what the consequences are if the mistake is not repaired. How are these forms of damage repaired? § Deamination- spontaneous loss of an amino group from a cytosine in DNA to produce the base uracil 19.Describe the repair process for DNA damage. What steps do repair of thymine dimers, depurination and deamination have in common? What steps are different? 1. the damaged DNA is recognized and removed by one of a variety of mechanisms. Involve nucleases, which cleave the covalent bonds that join the damaged nucleotides to the rest of the DNA strand, leaving a small gap on one strand of the DNA double helix in the region 2. a repair DNA polymerase binds to the 3’- hydroxyl end of the cut dna strand. It fills in the gap by making a complementary copy of the info stored in the undamaged strand 3. a break remains in the sugar-phosphate backbone of the repaired strand. This nick is sealed by DNA ligase, the same enzyme that joins the Okazaki fragments during replication of the lagging DNA strands. 20.When does mismatch repair occur? How does that compare to when the repair of thymine dimers, depurination, and deamination take place? § Mismatch- functions immediately after DNA synthesis § Depurination, deamination, and pyrimidine dimers- repairs DNA damage that occurs at other times in cell cycle § A complex of mismatch repair proteins recognizes such a DNA mismatch, removes a portion of the DNA strand containing the error, and then resynthesizes the missing DNA. This repair mechanism restores the correct sequence. § The mismatch system always removes a portion of the newly made DNA strand. Chapter 7 Objectives After completing chapter 7, you should be able to answer the following questions. 1. What is the central dogma? How does genetic ‘information’ flow through cells? § Unity of life- DNA replication, RNA synthesis, and protein synthesis- all cells follow these processes § When a particular protein is needed by the cell, the nucleotide sequence of the appropriate segment of a DNA molecule is first copied into another type of nucleic acid- RNA. That segment of DNA is called a gene, and the resulting RNA copies are then used to direct the synthesis of the protein. § The flow of genetic information in cells is from DNA to RNA to protein- all cells express their genetic information in this way- central dogma. § Cells copy DNA into RNA- transcription § Use the information in RNA to make protein- translation 2. Define the concept of gene expression. Are all genes expressed in at the same level in all cells? What is the first step in gene expression? § The production of RNA is the first step in gene expression- copy the nucleotide sequence of that gene into RNA- transcription- written in the language of nucleotides § Gene expression refers to the process by which the information encoded in a DNA sequence is translated into a product that has some effect on a cell or organism. When the final product is a protein, gene expression includes both transcription and translation. When RNA is the final product, gene expression doesn’t include translation. 3. What is the main enzyme that catalyzes transcription? Does it require a primer to function? § RNA polymerase- unwinds DNA, adds nucleotides, RNA is displaced immediately, DNA rewinds. Next RNA is started before the first RNA is complete § Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the strands of DNA then acts as a template for the synthesis of RNA. RNA is added to the growing rna chain. The incoming ribonucleotide is covalently linked to the growing RNA chain by the enzyme RNA polymerase. The RNA transcript is elongated one nucleotide at a time and has a nucleotide sequence exactly complementary to the strand of DNA used as the template § Behind where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms (why RNA is single stranded). § RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together and form the sugar-phosphate backbone of the RNA chain. § The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead to expose a new region of the template strand for complementary base-pairing. Goes from 5’ to 3’. § The incoming ribonucleoside triphosphates provide energy needed to drive the reaction forward. § RNA polymerase uses ribonucleoside for phosphates as substrates, so it catalyzes the linkage of ribonucleotides. RNA polymerase can start an RNA chain without a primer. § RNA is not used as the permanent storage form of genetic information in cells, so mistakes in RNA transcripts have relatively minor consequences for a cell 4. What sequences indicate where transcription should start and stop in bacteria? § RNA polymerase recognizes a promoter- conserved sequence- particular subunit- sigma. Promoter dictates which strand- where to make the RNA and how much RNA to make § When RNA polymerase collides randomly with a DNA molecule, the enzyme sticks weakly to the double helix and then slides rapidly along its length. RNA polymerase latches on tightly only after it has encountered a gene region called a promoter, which contains a specific sequence of nucleotides that lies immediately upstream of a starting point for RNA synthesis. § Once tightly bound to this sequence, the RNA polymerase opens up the double helix immediately in front of the promoter to expose the nucleotides on each strand of a short stretch of DNA. One strand of DNA acts as a template for base pairing for the incoming ribonucleoside triphosphates, 2 of which are joined by the polymerase to begin synthesis of the RNA chain. § This continues until the enzyme encounters the terminator, where the polymerase halts and releases both the DNA template and the newly made RNA transcript. § Every promoter has a certain polarity: it contains 2 different nucleotide sequences upstream of the transcriptional start site that position the RNA polymerase, ensuring that it binds to the promoter in only one orientation § Once the enzyme is bound, it must use the DNA strand oriented in the 3’ to 5’ direction as its template. 5. How is transcription initiated in eukaryotes? § Bacterial RNA polymerase can initiate transcription. Eukaryotic polymerase need accessory proteins-general transcription factors- assemble at the promotions § Initiation with general transcription factors- required at all promoters § TFIID binds to TATA box-causes dramatic shape change- landmark § Other proteins attracted- TFIIE, TFIH, RNA pol § General transcription factors come in, recruited by shape change, order of assembly probably varies § RNA Pol II must be released-phosphates added to RNA Pol II tail by a kinase in TFIIH. General transcription factors released § General transcription factors- assemble on the promoter, where they position the RNA polymerase and pull apart the DNA double helix to expose the template strand, allowing the polymerase to begin transcription. § The assembly process begins with the binding of the general transcription factor TFIID to a short segment of DNA double helix composed primarily of T and A nucleotides because of its composition- the TATA box. TFIID causes a distortion in the DNA double helix, which helps to serve as a landmark for the subsequent assembly of other proteins at the promoter. The TATA box is a key component of many promoters used by RNA polymerase II, and it located 25 nucleotides upstream from the transcription start site. § Once TFIID has bound to the TATA box, the other factors assemble, along with RNA polymerase II, to form a complete transcription initiation complex. § Once RNA polymerase II has been positioned on the promoter, it is released from the complex to begin making an RNA molecule. An addition of phosphates to its “tail”- initiated by the general transcription factor TFIIH, which contains a protein kinase as one of its subunits. Once transcription has begun, the general transcription factors dissociate from the DNA and then initiate another round of transcription with a new RNA polymerase molecule. § When RNA polymerase II finishes transcribing a gene, it too is released from the DNA- the phosphates are stripped off by protein phosphatases and the polymerase is then ready to find a new promoter. § Only the dephosphorylated form of RNA polymerase II can initiate RNA synthesis 6. What are three ways that RNA differs from DNA, with respect to structure? § How RNA and DNA differ physically: RNA is single stranded and DNA is double stranded § Because RNA is single stranded, it can fold up to form the final shape of a protein; DNA cannot fold in this fashion § RNA molecules are much shorter than DNA molecules 7. What are the 4 major types of RNA in the cell, and what are their functions in the cell? Types of RNA Function Messenger RNAs (mRNAs) Code for proteins Ribosomal RNAs (rRNAs) Form the core of the ribosome’s structure and catalyze protein synthesis microRNAs (miRNAs) Regulate gene expression Transfer RNAs (tRNAs) Serve as adaptors between mRNA and amino acids during protein synthesis Other noncoding RNAs Used in RNA splicing, gene regulation, telomere maintenance, and may other processes 8. What are three ways that eukaryotic mRNAs must be processed in the nucleus before they are translated in the cytosol? § Capping- 7- methylguanosine cap, occurs during transcription. Cap and tail- increases stability, aid in export from nucleus, and identify molecule as an RNA. Modifies the 5’ end of the RNA transcript, the end that is synthesized first. The RNA is capped by the addition of an atypical nucleotide. This capping occurs after RNA polymerase II has produced about 25 nucleotides of RNA. § Polyadenylation- provides a newly transcribed mRNA with a special structure at its 3’ end. The 3’ end of a forming eukaryotic mRNA is first trimmed by an enzyme that cuts the RNA chain at a particular sequence of nucleotides. The transcript is then finished off by a second enzyme that adds a series of A nucleotides to the cut end. § These two increase the stability, facilitate its export from the nucleus to the cytoplasm, and mark the RNA molecule as an mRNA. They are used by the protein synthesis machinery to make sure that both ends of the mRNA are present and that the message is complete before protein synthesis begins. § Splicing § Capping and polyadenylation occur only on RNA transcripts destined to become mRNA molecules. 9. What are introns and exons? § Exons- expressed sequences § Introns- intervening sequences. Most human genes have introns. Introns are frequently larger than exons. Specific nucleotide sequences are present at the start and end of introns. § Most protein-coding eukaryotic genes, have their coding sequences interrupted by long, noncoding intervening sequences called introns. The scattered pieces of coding sequence-called expressed sequence or exons- are shorter and represent only a small fraction of the total length of the gene § To produce an mRNA in a eukaryotic cell, the entire length of the gene, introns as well as exons is transcribed into RNA. 10.What is RNA splicing, and why is it important? 1) Adenine in intron sequence attacks the backbone 2) The free end of the intron becomes covalently attached 3) The 3’ end of the exon reacts with the start of the next exon 4) Exons are joined 5) Intron is released as a ‘lariat’ structure § Splicing catalyzed by an RNA/ protein machine- spliceosome § RNAs: small, nuclear RNAs (snRNAs)- with proteins, form: small nuclear ribonucleoprotein § snRNps § snRNPs form core of spliceosome § catalytic • after capping and during RNA Polymerase II transcribing the gene, the process of splicing begins. Introns are removed from the newly synthesized RNA and the exons are stitched together. • Once a transcript has been spliced and its 5 and 3 ends have been modified, the RNA is now functional mRNA molecule that can leave the nucleus and be translated into protein. • Each intron contains a few short nucleotide sequences that act as cues for its removal from pre-mRNA. Guided by these sequences, a splicing machine cuts out the intron in the form of a lariat structure. • RNA splicing is carried out by RNA molecules, called small nuclear RNAs (snRNAs), packaged with additional proteins to form small nuclear ribonucleoproteins (snRNPs). They recognize splice-site sequences through complementary base-pairing between their RNA components and the sequences in the pre-mRNA. These snRNPs form the core of the spliceosome- the large assembly of RNS and protein molecules that carries out RNA splicing in the nucleus. 11.What would happen if an intron was not precisely spliced out of an RNA? The reading sequence would not be correct and it would alter the sequence of amino acids 12.What are the consequences of mistakes made during DNA replication versus mistakes made during transcription? Which would be more severe? Any mistakes made during DNA replication is going to affect all the RNA and protein is made from that sequence. Mistakes in transcription would affect that specific RNA strand and those proteins. 13.Where does the process of translation take place in the cell? In the ribosomes, cytosol. 14.What is a codon? How is the genetic code translated into a specific amino acid? § Code of mRNA is read in groups of three nucleotides, or codons. Genetic code is how the nucleotide sequence of a gene is converted into amino acids § Each group of three consecutive nucleotides in RNA is called a codon, and each codon specifies one amino acid. 15.How many possible codons are there? How many possible amino acids? Is one amino acid specified by more than one codon? § 64 total possible codons § 20 possible amino acids 16.What is tRNA charging, and why is it important that it be done correctly? § Amino-acyl tRNA synthetase connects amino acid with its matching tRNA. 20 synthetases, one for each amino acid § ATP is hydrolyzed to AMP § The codons in an mRNA molecule do not directly recognize the amino acids they specify, rather, the translation of mRNA into protein depends on adaptor molecules that can recognize and bind to a codon at one site on their surface and to an amino acid at another site. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs). 17.What is the role of tRNA in protein synthesis? § tRNA- the adaptor molecule- transports amino acids and holds them into place on the ribosome in their incorporation into proteins. It reads the mRNA code, bringing in the correct amino acid. § tRNAs can fold into double helical structure § Two regions of unpaired nucleotides situated at either end of the L-shaped tRNA molecule are crucial to the function of tRNAs in protein synthesis. One of these regions forms the anticodon, a set of three nucleotides that bind through base-pairing, to the complementary codon in an mRNA molecule. The other is a short single-stranded region at the 3’ end of the molecule; this is the site where the amino acid that matches the codon is covalently attached to the tRNA. § There is more than one tRNA for many of the amino acids, and some tRNA molecules can base-pair with more than one codon. Some tRNAs are constructed so that they require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch or wobble at the third position. § Wobble base-pairing make it possible to fit the 20 amino acids to their 61 codons with as few as 31 kinds of tRNA molecules. § tRNA must be linked or charged with the correct amino acid. Recognition and attachment of the correct amino acid depend on enzymes called aminoacyl- tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules. It is the combined action of synthetases and tRNAs that allows each codon in the mRNA molecule to specify its proper amino acid. § The synthetase-catalyzed reaction that attaches the amino acid to the 3’ end of the tRNA is one of many reactions in cells coupled to the energy releasing hydrolysis of ATP. § The ribosome is a large complex made from dozens of small proteins and several crucial RNA molecules called ribosomal RNAs (rRNAs). They can move along the mRNA, capture complementary tRNA molecules, hold the tRNAs in position, and then covalently link the amino acids that they carry to form a polypeptide chain. § Ribosomes have a large subunit and small subunit: the small matches the tRNAs to the codons of the mRNAs, while the large catalyzes the formation of the peptide bonds that covalently link the amino acids together into a polypeptide chain. They come together on an mRNA molecule near its 5’ end to start the synthesis of a protein. The mRNA is pulled through the ribosome like a piece of tape. As the mRNA goes from 5’ to 3’, the ribosome translates its nucleotide sequence into amino acid sequence, using tRNAs as adaptors. When synthesis of the protein is finished, the 2 subunits of the ribosome separate. § In addition to a binding site for an mRNA molecule, each ribosome contains 3 binding sites for tRNA molecules: A, P and E. § To add an amino acid: the charged tRNA enters the A site by base pairing with the codon on the mRNA molecule. Its amino acid is then linked to the peptide chain held by the tRNA in the neighboring P site. Next, the large ribosomal subunit shifts forward, moving the spent tRNA to the E site before ejecting it. 18.How (and where, on the RNA) is protein synthesis initiated? What roles do initiation factors play? • The translation of an mRNA begins with the codon AUG, and a special charged tRNA is required to initiate translation. This initiator tRNA always carries the amino acid methionine. All newly made proteins all have methionine as the first amino acid at their N-terminal end. • In eukaryotes, an initiator tRNA, charged with methionine, is first loaded into the P site of the small ribosomal subunit, along with additional proteins, called translation initiation factors. Only a charged initiator tRNA molecule is capable of binding tightly to the P site in the absence of the large ribosomal subunit. The small ribosomal subunit loaded with the initiator tRNA binds to the 5’ end of an mRNA molecule, which is marked by the 5’ cap that is present on all eukaryotic mRNAs. The small ribosomal subunit then moves forward (5’ to 3’) along the mRNA searching for the first AUG. When this AUG is encountered and recognized by the initiator tRNA, several initiation factors dissociate from the small ribosomal subunit to make way for the large ribosomal subunit to bind and complete ribosomal assembly. • For bacteria- mRNAs have no caps, they contain specific ribosome-binding sequences. A prokaryotic ribosome can readily bind directly to a start codon that lies in the interior of an mRNA, as long as a ribosome-binding sequences site precedes it by several nucleotides. • mRNAs are polycistronic- they encode several different proteins, each of which is translated from the same mRNA molecule. • The end of translation is signaled by the presence of one of several codons- UAA, UAG, and UGA. • Proteins known as release factors bind to any stop codon that reaches the A site on the ribosome; this binding alters the activity of the peptidyl transferase in the ribosome, causing it to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA. This reaction frees the carboxyl end of the polypeptide chain from its attachment to a tRNA molecule; because this is the only attachment that holds the growing polypeptide to the ribosome, the completed protein chain is immediately released. At this point, the ribosome also releases the mRNA and dissociates into its two separate subunits. 19.Compare and contrast initiation of translation in prokaryotes and eukaryotes. What AUG is used in each? § Prokaryotic mRNAs are polycistronic- encode more than one protein, ribosome binding sites, AUG in context § Eukaryotic mRNAs- small subunit binds to cap, slides down to first AUG 20.Why can’t the first AUG in the mRNA be used to start translation in prokaryotes? • A prokaryotic ribosome can readily bind directly to a start codon that lies in the interior of an mRNA. 21.How is translation terminated? § 3 codons: stop codons- UAA, UAG, and UGA. § No complementary tRNA § Requires release factors, or proteins that resemble tRNAs § No corresponding amino acid § GTP is hydrolyzed § Polypeptide reacts with water, released from P site § The end of translation is signaled by the presence of one of several codons- UAA, UAG, and UGA. § Proteins known as release factors bind to any stop codon that reaches the A site on the ribosome; this binding alters the activity of the peptidyl transferase in the ribosome, causing it to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA. This reaction frees the carboxyl end of the polypeptide chain from its attachment to a tRNA molecule; because this is the only attachment that holds the growing polypeptide to the ribosome, the completed protein chain is immediately released. At this point, the ribosome also releases the mRNA and dissociates into its two separate subunits. 22.What is the catalytic molecule in a ribosome? § Polyribosomes- complex of mRNA and multiple ribosomes § ribozymes 23.What is protein degradation, and how can it be controlled in the cell? § Controlled breakdown also helps regulate activity in cell § Proteases break down proteins § Proteasome operates in the cytosol, on proteins with ubiquitin “tags” § Cells possess specialized pathways that enzymatically break proteins down into their constituent amino acids. The enzyme that degrades proteins, first to short peptides and finally to individual amino acids- proteases. § Proteases act by cutting the peptide bonds between amino acids. § In eukaryotic cells, proteins are broken down by large protein machines, called proteasomes, present in both the cytosol and the nucleus. A proteasome contains a central cylinder formed from proteases whose active sites face into an inner chamber. Each end of cylinder is stoppered by a large protein complex formed from at least 10 types of protein subunits. These protein stoppers bind the proteins destined for degradation and then, using ATP hydrolysis to fuel this activity, unfold the doomed proteins and thread them into the inner chamber of the cylinder. Proteases chop them into short peptides, which are then jettisoned from either end of the proteasome. § Proteasomes act primarily on proteins that have been marked for destruction by the covalent attachment of a small protein called ubiquitin. Specialized enzymes tag selected proteins with a short chain of ubiquitin molecules; these ubiquitylated proteins are then recognized, unfolded, and fed into proteasomes by proteins in the stopper 24.What are three ways that transcription and translation vary between prokaryotes and eukaryotes? Bio214 Chapter 11 Objectives 1. How does the cell membrane create barriers that confine particular molecules to specific compartments? 2. Describe the construction of a typical cell membrane. The important components are the proteins and the lipids. Hydrophilic heads and hydrophobic tails- amphipathic 3. What does it mean that membrane lipid molecules are amphipathic? Hydrophobic tails and hydrophilic heads 4. Why do membranes spontaneously assemble into bilayers, and then into sealed compartments? Amphipathic molecules, such as phospholipids are subject to two conflicting forces: the hydrophilic head is attracted water, while the hydrophobic tails shun water and seek to aggregate with other hydrophobic molecules, which is resolved by the formation of the lipid bilayer. It is energetically favorable. When there is a tear, the hydrophobic tails are faced with water, which energetically unfavorable, so the molecules of the bilayer will spontaneously rearrange to eliminate the free edge. 5. Describe the construction of a phospholipid. Phosphate + glycerol + fatty acids. R is a variable polar head group 6. Describe the construction of a sphingolipid. Amino alcohol + hydrocarbon chain 7. Describe cholesterol. How does it compare to other classes of membrane lipids? It is smaller in size, stiffer. Present in the plasma membrane. Constitutes approximately 20% of the lipids in the membrane by weight. They are short and rigid, they fill the spaces between neighboring phospholipid molecules left by the kinks in their unsaturated hydrocarbon tails. Stiffens the bilayer, making it less flexible and less permeable. 8. How can lipids move in membranes? Are they restricted to a specific position? What types of motion do they undergo? Membranes move. Types of motion: diffusion within membrane, rotation, flexion, flip-flop. Fluid-mosaic model: lipid is a fluid with a mosaic of discontinuous protein particles. Some types of movements are rare, while others are frequent and rapid. In synthetic lipid bilayer phospholipid molecules very rarely tumble from one half of the bilayer, or monolayer to the other. Without proteins to facilitate this process, flip-flop occurs. Lipid molecules continuously exchange places with their neighbors in the same monolayer. Individual lipid molecules not only flex their hydrocarbon tails, but they also rotate rapidly about their long axis. 9. Are the two layers of a lipid bilayer identical? Why might the two layers have different compositions? Most cell membranes are asymmetrical: the two halves of the bilayer often include different sets of phospholipids. New phospholipids are manufactured by enzymes bound to the cytosolic surface of the ER. Using free fatty acids as substrates, the enzyme deposit the newly made phospholipids exclusively in the cytosolic half of the bilayer. The lipids are catalyzed by enzymes called scramblases, which remove randomly selected phospholipids from one half of the lipid bilayer and insert them into the other. 10.How is the asymmetry of the lipid bilayer generated? Arrangement is energetically most favorable. Hydrophilic groups can interact with water, hydrophobic groups protected from aqueous environment, minimizes ordering of water molecules. Begins in the golgi apparatus. Flippases, a phospholipid- handling enzyme remove specific phospholipids from the side of the bilayer facing the exterior space and flip them into the monolayer that faces the cytosol. The cytosolic monolayer always faces the cytosol, while the noncytosolic monolayer is exposed to either the cell exterior, or to the interior space of an organelle. 11.What is membrane fluidity? Why is it important? Fluidity is how easily lipid molecules move in the membrane. Compromise between need for mobility and the need for structure and support. Allows interactions to take place in the membrane. The closer and more regular the packing of the tails, the more viscous and less fluid the bilayer will be. Two properties of hydrocarbon tails that affect how tightly they pack in the bilayer: their length and the number of double they contain. Double bonds= unsaturated. Single bond= saturated. Fluidity enables many membrane proteins to diffuse rapidly in the plane of the bilayer and to interact with one another. It permits membrane lipids and proteins to diffuse from sites where they are inserted into the bilayer after their synthesis to other regions of the cell. It ensures that membrane molecules are distributed evenly between daughter cells when a cell divides. It allows membranes to fuse with one another and mix their molecules. 12.How do cells increase their membrane fluidity? List three ways. Unsaturated fatty acids increase membrane fluidity. Remove cholesterol. Changing single bonds to double bonds. Reshuffling fatty acid chains- two unsaturated fatty acids together. 13.Describe how bacterial transformation exploits changes in membrane fluidity. 14.What type of protein structure (2°) typically crosses a membrane? Why is this type of transmembrane domain so common? For a transmembrane receptor protein: the part of the protein that receives a signal from the environment must be on the outside of the cell, whereas the part that passes along the signal must be in the cytosol. This orientation is a consequence of the way in which membrane proteins are synthesized. The portions of a transmembrane protein located on either side of the lipid bilayer are connected by specialized membrane-spanning segments of the polypeptide chain. These segments, which run through the hydrophobic environment of the interior of the lipid bilayer, are composed of amino acids with hydrophobic side chains. However, the peptide bonds that join the successive amino acids in a protein are normally polar, making the polypeptide backbone hydrophilic. Hydrogen bonding is maximized if the polypeptide chain forms a regular a helix, and so the great majority of the membrane spanning segments of polypeptide chains transverse the bilayer as a helices. 15.If a membrane protein has one transmembrane domain, is this domain likely to be hydrophobic, hydrophilic or amphipathic? Why? hydrophobic 16.If five transmembrane domains come together to form an ion channel, are these α-helices likely to be hydrophobic, hydrophilic, or amphipathic? Why? In many of these multipass transmembrane proteins, one or more of the membrane spanning regions are amphipathic-formed from a helices that contain both hydrophobic and hydrophilic amino acid side chains. 17.What is the difference between integral membrane proteins and peripheral membrane proteins? Transmembrane: one or more membrane-spanning domains- also called integral membrane protein. Lipid-linked- covalently linked to a membrane lipid or fatty acid- integral membrane protein. Protein attached: noncovalent interactions with integral membrane protein or lipids-peripheral Proteins that are directly attached to the lipid bilayer (transmembrane, associated with the lipid monolayer, or lipid-linked) can be removed only by disrupting the bilayer with detergents. These proteins are integral membrane proteins. The remaining membrane proteins are peripheral membrane proteins, they can be released from the membrane by more gentle extraction procedures that interfere with protein-protein interactions but leave the bilayer intact. 18.Membrane proteins are frequently glycosylated. What does this mean, and what function does glycosylation serve? Majority of proteins in plasma membrane have short chains of sugars, called oligosaccharides linked to them, called glycoproteins. All of the carbohydrate on the glycoproteins, proteoglycans, and glycolipids is located on the outside of the plasma membrane, where it forms a sugar coating called the carbohydrate layer or glycocalyx. Helps protect the cell surface from mechanical damage. They absorb water and give the cell a slimy surface, which helps motile cells squeeze through narrow spaces and prevents blood cells from sticking to one another or to the walls of blood vessels. Have an important role in cell-cell recognition and adhesion. 19.Cell membranes are fragile. How can they be reinforced? Cell cortex: a meshwork of fibrous proteins- main component is spectrin (red fibers). Connected to membrane through transmembrane proteins. Connected to the cytoskeleton. Most cell membranes are strengthened and supported by a framework of proteins, attached to the membrane via transmembrane proteins. For plants, yeast and bacteria, the cell’s shape and mechanical properties are conferred by a rigid cell wall- a mesh work of proteins, sugars, and other macromolecules that encases the plasma membrane. The plasma membrane of animal cells is stabilized by a meshwork of fibrous proteins, called the cell cortex that is attached to the underside of the membrane. The main component of the cortex is the dimeric protein spectrin. Forms a meshwork that provides support for the plasma membrane and maintains the cell’s biconcave shape--- in blood cells. In animal cells, the cortex is rich in actin and myosin, need the cortex to allow them to selectively take up material from their environment, change their shape actively, and to move. Also use their cortex to restrain the diffusion of proteins within the plasma membrane. 20.How can cells restrict movements of proteins in the plasma membrane? Describe the methods. Tight junctions restrict movement of protein A. Specialized proteins form a continuous barrier around the cell, creating a seal between adjacent membranes. Cells have ways of confining particular proteins to localized areas within the bilayer membrane, creating functionally specialized regions or membrane domains on the cell or organelle surface. Tight junction—specialized junctional proteins form a continuous belt around the cell where the cell contacts its neighbors, creating a seal between adjacent plasma membranes. BIO214 Chapter 12 Objectives After completing Chapter 12, you should be able to answer the following questions: 1. What types of molecules is a lipid bilayer (with no proteins!) permeable to? What types of molecules is the lipid bilayer not permeable to? Small molecules move more easily, less polar molecules- cross more easily. Membrane is impermeable to ions- charge interacts with water. Small nonpolar molecules can go through— O2, CO2, N2, steroid hormones. Small uncharged polar molecules can but not as well—H2O, ethanol, glycerol. Larger uncharged polar molecules can but not as well- amino acids, glucose, nucleosides. Ions cant- H+, Na+, K+, Ca2+ 2. How are molecules that cannot cross a lipid bilayer brought into the cell? Specialized membrane transport proteins that span the lipid bilayer, providing private passageways across the membrane for select substances. 3. What are the two classes
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