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UF / Biology / BIOL 3603 / who demonstrated that dna is the genetic material of the t2 phage

who demonstrated that dna is the genetic material of the t2 phage

who demonstrated that dna is the genetic material of the t2 phage

Description

School: University of Florida
Department: Biology
Course: Genetics
Professor: W. barbazuk
Term: Fall 2016
Tags:
Cost: 50
Name: PCB3063: Exam 2
Description: All the notes for chapters 10-17, what will be covered on exam 2 on 11/01
Uploaded: 10/31/2016
42 Pages 180 Views 1 Unlocks
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• How does the repair system recognize which strand is correct?




- How do spontaneous mutations arise?




- Salvador Luria and Max Delbruck asked this question: Can organisms some how ‘select’ or ‘direct’ the mutation of genes in order to adapt to a particular environmental pressure?



Chapter 10 DNA Structure and Analysis - The genetic material must exhibit four characteristics; It must be able to replicate,  store information, allow variation by mutation, express If you want to learn more check out soc gen 5 ucla
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information - Central Dogma of Molecular Genetics: “DNA makes RNA, which makes proteins” - In the mid 1900s – it was not clear which molecule was the “genetic material”; the  genetic material is physically transmitted from parent to offspring; proteins and nucleic  acids were the major candidates for the genetic material - For a long time, protein was favored to be the genetic material; it is abundant in cells;  it was the subject of the most active areas of genetic research - DNA was thought to be too simple to be the genetic material, with only four types of nucleotides as compared to the 20  different amino acids of proteins - Evidence in favor of DNA being the  genetic material came for studies  of bacteria and phages; bacteria:  transformation (Griffith, then Avery  McLeod and McCarty; phage:  Hershey and Chase) - Transformation studies I (right) -  Griffith: Griffith showed that  avirulent strains of Steptococcus  pneumoniae could be transformed  to virulence; speculated that the  transforming principle could be part  of the polysaccharide capsule or  some compound required for  capsule synthesis • Transformation is a heritable  change in a cell or an organism  brought about by exogenous  DNA (heat-killed bacteria in this  case)- Transformation studies II (Avery, McLeod and McCarty) (right) - Phage studies: Hershey and Chase  (below) : T2 phage consists of 50% protein  and 50% DNA. Infection is initiated by  adsorption of the phage by its tail fibers to  the bacteria. Production of new virus  occurs in the cell • lytic cycle: new phages are constructed  and the bacterial cell is lysed, releasing  the progeny viruses  - Protoplasts (or spheroplasts) are  enzymatically treated cells; contain only the  cell membrane as their outer boundary - Transfection: process of infection by only the viral nucleic acid - Tetranucleotide hypothesis: Levene postulated incorrectly that identical groups of four  nucleotides were repeated - Ribonuclease (RNase): enzymes that  hydrolyze RNA - Deoxyribonuclease (DNase): DNA digesting enzyme  - Nucleotides are the building blocks of  DNA; They consist of: a nitrogenous base;  a pentose sugar; a phosphate group - The nitrogenous bases can be purines or  pyrimidines; The purines are adenine (A) and guanine (G); The pyrimidines are  cytosine (C), thymine (T), and uracil (U) - RNA contains ribose as its sugar; DNA  contains deoxyribose (one less oxygen) - A nucleoside contains the nitrogenous base and the pentose sugar - A nucleotide is a nucleoside with a phosphate group added - The C-1' position is the location of the nitrogenous base on a  nucleotide/nucleoside; the C-5' position is the location of the  phosphate group on a nucleotide. - Nucleotides can have one, two, or three phosphate groups  and are called NMPs, NDPs, and NTPs, respectively  - Nucleotides are linked by a phosphodiester bond between  the phosphate group at the C-5' position and the OH group  on the C-3' position. - BASE COMPOSITION: Chargaff showed that the amount of A is proportional to T  and the amount of C is proportional to G, but the percentage of C + G does not  necessarily equal the percentage of A + T - X-RAY diffraction: showed a 3.4 angstrom periodicity, characteristic of a helical  structure - Watson and Crick Model: 2 polynucleotide chains coiled around a central axis – form  a right hand double helix; major groove alternates with a minor  groove; diameter of helix is 20 A (2nm); Chains are anti parallel; Each turn is 34 A (3.4nm) = 10 bases; Bases of  opposite chains are paired. H-bonds between A and T, C and  G; Bases are flat, lie perpendicular to the axis, and ‘stack’;  The A-T and G-C base pairing provides complementarity of  the two strands and chemical stability to the helix; A-T base  pairs form two hydrogen bonds and G-C base pairs form  three hydrogen bonds  - The Watson-Crick DNA model is of  B-DNA, which is believed to be the biologically significant  form; A-DNA is slightly more compact than  B-DNA; C-DNA, D-DNA, and E-DNA are also right-handed  forms of DNA that are less compact than B-DNA. - RNA structure similar to DNA but single stranded: • the sugar ribose replaces deoxyribose of DNA  • uracil replaces thymine of DNA • Most RNA is single stranded, although some RNAs form double-stranded regions  as they fold into different secondary structures- There are three classes of cellular RNAs:  • messenger RNA (mRNA): templates for protein synthesis • ribosomal RNA (rRNA): components of ribosomes • transfer RNA (tRNA): carry amino acids for protein synthesis - Nucleic acids absorb UV light most strongly at 260 nm due to interaction between UV  light and the ring systems of the bases; Therefore, UV light can be used in the  localization, isolation and characterization of molecules that contain nitrogenous  bases; UV analysis is used in conjunction with many procedures that separate and  identify molecules - Sedimentation: Nucleic acids can be separated by gradient centrifugation  procedures; Sedimentation equilibrium centrifugation separates by density gradient. - Sedimentation velocity centrifugation used UV optics during sedimentation to  measure the velocity of sedimentation in Svedberg coefficient units. Key variables are  mass and shape of the molecule under study - Denaturation and Renaturation of Nucleic Acids: Hyperchromic shift during DNA  denaturation is used to determine the melting temperature (Tm); As DNA melts  (denatures) both the UV absorption and buoyant density increases.  - Reassociation kinetics provides information about the size and complexity of  genomic DNA from an organism; DNA is fragmented, melted and allowed to renature  while monitoring absorbance; Plot the percentage of reassociated DNA vs. a  logarithmic scale of normalized time • C/Co = 1/1+kCot • k is second order rate constant; t is time, Co = initial concentration of single stranded DNA; C is concentration of single-stranded DNA after time t has elapsed.  - If all pairs of single-stranded complements include unique nucleotide sequences and  are of similar size, the half-reaction time varies directly with complexity (X);  Represents the total length of in nucleotide pairs of all unique DNA fragments laid end  to end - In molecular hybridization, DNA strands (or DNA and RNA strands) can be  renatured to each other. - Fluorescent in situ hybridization (FISH) is used to identifying the chromosomal  location of a DNA of interest. - Nucleic Acid Electrophoresis separates DNA and RNA  fragments by size such that smaller fragments migrate  through a gel at a faster rate than large fragments  Chapter 11 Chapter 11: DNA Replication and  Recombination - The complementarity of DNA strands allows each  strand to serve as a template for synthesis of the other - Three possible modes of DNA replication are possible: • conservative  • semiconservative • dispersive  - The Meselson-Stahl experiment demonstrated that DNA replication is semi  conservative; each new DNA molecule consists of one old strand and one newly  synthesized strand - Taylor-Woods-Hughes experiment demonstrated semi conservative replication in  eukaryotes; used root tips of Vicia fabia (excellent source of dividing cells); monitored  replication by labeling with H3-thymidine (radioactive) - DNA Replication in Bacteria: Originates at ‘origins of replication’ in bacterial cells;  strands unwind during replication creating a ‘replication fork’; replication is  bidirectional; segment of DNA that is replicated is a ‘replicon’ – in bacteria this is the  whole chromosome - DNA Synthesis in Bacteria involves five polymerases, as well as other enzymes - Chain elongation occurs in the 5' to 3' direction by addition of one nucleotide at a time  to the 3' end. - As the nucleotide is added, the two terminal phosphates are cleaved off  - DNA polymerases I, II, and III can elongate an existing DNA strand (called a primer)  but cannot initiate DNA synthesis; All three possess 3' to 5' exonuclease activity; but  only DNA polymerase I demonstrates 5' to 3' exonuclease activity.- DNA polymerase III is the enzyme  responsible for the 5' to 3'  polymerization essential in vivo; Its  3' to 5' exonuclease activity allows  proofreading.  - Polymerase I is believed to be  responsible for removing the  primer and the synthesis that fills  gaps produced during synthesis - DNA polymerases I, II, IV, and V  are involved in various aspects of  repair of damaged DNA - DNA polymerase III is a holoenzyme composed of 10 subunits: (right) - There are seven key issues that must be resolved during DNA replication: • unwinding of the helix  • reducing increased coiling generated during unwinding  • synthesis of a primer for initiation  • discontinuous synthesis of the second strand • removal of the RNA primers • joining of the gap-filling DNA to the adjacent strand • proofreading - Step 1: open helix and stabilize (right) • DnaA binds to the origin of replication and is responsible  for the initial steps in unwinding the helix • Subsequent binding of DnaB and DnaC further opens  and destabilizes the helix • Single-stranded binding proteins (SSBPs) stabilize the  open conformation • Proteins such as these, which require the energy  normally supplied by the hydrolysis of ATP to break  hydrogen bonds and denature the double helix, are called helicases- Step 2: release tension created by unwinding DNA at replication fork • Unwinding produces supercoiling that is relieved by DNA gyrase, a member of a  larger group of enzymes referred to as DNA topoisomerases. - Step 3: primer synthesis • DNA PolIII needs a primer with a free 3’ –OH to add bases too. • Primase directs the synthesis of a small 10-12nt primer - Step 4: Pol III synthesizes DNA • Synthesis occurs on both strands simultaneously. The net effect is that the PolIII  holoenzyme moves away from the origin. • DNA synthesis by PolIII occurs only in the 5’->3’ direction • Recall that the DNA strands are antiparallel – therefore synthesis must occur in one  direction on one strand, and in the opposite direction on the other. • As the replication fork moves, one strand serves as a template for continuous DNA  synthesis (leading strand). The opposite strand undergoes discontinuous synthesis  (lagging strand). • The lagging strand is synthesized as Okazaki fragments, each with an RNA  primer. - Step 5 and 6: primer removal and gap repair • DNA polymerase I removes the primers on the lagging strand and the fragments  are joined by DNA ligase - Concurrent synthesis of the leading and lagging strands. • Both DNA strands are synthesized concurrently by looping the lagging strand to  invert the physical but not biological direction of synthesis.  - Proofreading and error correction • Base addition is not perfect. • All of the DNA polymerases have 3' to 5' exonuclease activity that allows  proofreading. • Mismatched bases can be detected and excised in the 3-5> direction. Once  removed, synthesis can proceed in the 5->3 direction.• This proofreading increases fidelity by a factor of 100. - Replication in Prokaryotes is controlled by a variety of genes; much of what is know  about replication in prokaryotes is based on genetic analysis of the process. - Eukaryotic DNA Synthesis Is Similar to Synthesis in Prokaryotes, but More Complex • Similar to bacterial process: dsDNA unwound at replication origin, replication forks  formed, synthesis is bidirectional creating leading and lagging strands. • Issues regarding eukaryotic cells:  - there is more DNA than prokaryotic cells  - the chromosomes are linear - the DNA is complexed with proteins • This makes DNA replication more complex in eukaryotes than in bacteria. • DNA synthesis is much slower. • Genomes are much larger. • Eukaryotic chromosomes contain multiple origins of replication to allow the genome  to be replicated in a few hours.  - Replication origins in Eukaryotes • Yeast autonomously replicating sequences (ARSs) contain an 11-bp consensus  sequence flanked by other short sequences involved in efficient initiation • Clusters of 20-80 ARSs are activated sequentially during S phase • The ARSs are initially bound by a group of proteins to form the origin recognition  complex (ORC). This occurs during G1 of the cell cycle • When kinases are bound along with the ORC a prereplication complex is formed  that is accessible to DNA polymerase • After the kinases are activated they serve to complete the initiation complex  directing localized unwinding and triggering DNA synthesis • Activation also inhibits reformation of the prereplication complex once DNA  synthesis has been completed at each replicon - Eukaryotic polymerases• Many DNA polymerases discovered, three are involved in replication of nuclear  DNA. • One involves mitochondrial DNA replication. • Others are involved in repair processes • Topology of the DNA helix has to change in order to be accessible to polymerase. • At each origin of replication synthesis begins with strand separation at AT rich  regions – this allows access by helicase • Histones must be dissociated, and they reassociate with the newly formed  duplexes. • Pol and d are the major forms of the enzyme involved in initiation and elongation. • 2 of the 4 Pol subunits act to synthesis the RNA primer. Pol possesses low  processivity, a term that reflects the length of DNA that is synthesized by an  enzyme before it dissociates from the template. After synthesizing 10nt of RNA  another subunit extends is by adding ~30 dNTPs.  • “Polymerase switching” occurs after the primer is in place (Pol dissociates from the  template and is replaced by d & e). Pol has high processivity and extends the  leading and lagging strands, as well as proofreads in the 3->5 direction.  - Telomeres protect chromosome ends • Telomeres at the ends of linear chromosomes consist of long stretches of short  repeating sequences and preserve the integrity and stability of chromosomes.  Necessary because the ds ends of the chromosome look like double-stranded  breaks. DSBs are one form of chromosomal damage – they can fuse with other  DSBs or become degraded by nucleases • Telomeres are composed of repeated 6bp units (TTGGGG / AACCCC or TTAGGG /  AATCCC). The G rich strand off (3’) has several hundred additional tandem copies  of the G rich sequence of the helix.  • folds back on itself forming G-quartets - Lagging strand synthesis at the end of the chromosome is a problem because once  the RNA primer is removed, there is no free 3'-hydroxyl group from which to elongate. - Telomere replication • Telomerase directs synthesis of the telomere repeat sequence to fill the gap• This enzyme is a ribonucleoprotein with an RNA that serves as the template for the  synthesis of its DNA complement. - Genetic recombination involves: • endonuclease nicking at an identical position in two homologs • strand displacement • ligation • branch migration • duplex separation to generate the characteristic Holliday structure - Genetic exchange at equivalent positions along two chromosomes with substantial  DNA sequence homology is referred to as general, or homologous, recombination. - The RecA protein in E. coli promotes the exchange of reciprocal single-stranded  DNA molecules. RecB, C and D involved in nicking and unwinding - Gene conversion is characterized by nonreciprocal genetic exchange between two  closely linked genes Chapter 12 DNA Organization into Chromosomes - Bacterial and viral genomes are relatively simple genomes – circular or liner nucleic  acid molecule; largely devoid of proteins; however, genomes are still large compared  to bacterial cell or virus capsule size - Bacterial chromosomes are compacted into a structure called a nucleoid - Bacterial DNA is associated with several DNA binding proteins – 2 are Hu and H1 • Small positively charged proteins that can bond ionically to the phosphate  backbone; similar to histones, but not actually involved in compacting DNA. - Bacterial chromosomes are compacted by supercoiling • Can observe 3 distinct components when centrifuging bacterial DNA • All 3 have same molecular weight • each with different sedimentation velocities that indicates different densities and  compactness• Supercoiling caused by underwinding DNA - Supercoils formed in the direction opposite to the unwinding. In this case the  supercoils are negative – they are left handed whereas the DNA helix is right handed - 2 identical molecules that differ only in linking number are Topoisomers – produced  by Topoisomerases cut one or both DNA strands and wind or unwind the helix  before resealing the ends - Specialized chromosomes reveal variations in the organization of DNA • Polytene chromosomes and lampbrush chromosomes are very large and can  be visualized by light microscopy. • Polytene chromosomes:  - have distinctive banding (chromomere) patterns - represent paired homologs (1000-5000) - are composed of many DNA strands - The DNA of the paired homologs of polytene chromosomes undergoes many rounds  of replication without strand separation or cytoplasmic division. - Polytene chromosomes have puff regions where the DNA has uncoiled and are  visible manifestations of a high level of gene activity. - Lampbrush chromosomes are large and have extensive DNA looping; they are found  in oocytes in meiotic prophase I (diplotene) - DNA organization into Chromatin • Chromatin – refer to uncoiled DNA present in interphase. • Re-entry into mitosis results in a 10,000 fold condensation (haploid genome ~6ft) • DNA in complexed with proteins – histones and non-histones • 5 types: H1, H2A, H2B, H3, H4 - Observations leading to current model of chromatin structure: • Digest chromatin with nucleases results in 200bp DNA fragments. - Chromatin consists of some repeating unit that protects DNA from cleavage. • Electron micrographs suggest chromatin fibers are linear arrays of spherical  particles.• Biochemical studies indicate that H2A, H2B, H3 and H4 occur as two types of  tetramers: (H2A)2 – (H2b)2 AND (H3)2- (H4)2; Repeating unit consists of one of each  tetramer – so histone ‘units’ are octomers • Long digestion times result in further reduction of the 200bp repeating unit – results  in 147bp; this is referred to as the nucleosome core particle (histone octamer  wrapped around 147 bp of DNA, histone H1 acts as a linker bound to the regions  between core particles - Chromatin remodeling must occur to allow the DNA to be accessed by DNA binding  proteins • Histone tails are important for histone modifications such as acetylation,  methylation, and phosphorylation • Acetylation opens up chromatin fibers • Methylation is associated with increase in gene activity • Phosphorylation is seen at characteristic times throughout the cell cycle - The chromosome is not structurally uniform; some parts of the interphase  chromosomes are condensed and can ‘stain, while other regions remain open - Euchromatin is uncoiled and active, whereas heterochromatin remains condensed  and is inactive - Mitotic chromosomes have a characteristic banding pattern • In C-banding, only the centromeres are stained • G-banding is due to differential staining along the length of each chromosome - Eukaryotic chromosomes demonstrate complex organization characterized by  repetitive DNA; ’Unique sequence’ constitutes only a small fraction of the genome real  estate – genes even less. - There is an abundance of repetitive elements • Consider heterochromatin associated with centromeres and telomeres. • Tandem repeats of short and long sequences • Transposable elements - Satellite DNA (~5% of human genome) is highly repetitive and consists of short  repeated sequences- Centromeres are the primary constrictions along eukaryotic chromosomes; mediate  chromosomal migration during mitosis and meiosis; In humans, alphoid family DNA  (~1M copies of 171bp sequence). - Telomeric DNA sequences consist of short tandem repeats that contribute to the  stability and integrity of the chromosome (~1000 copies).  - Middle repetitive sequences • Recognized by C0t analysis. • Most abundant are Variable number tandem repeats (VNTR). • 15-100bp long, found within and between genes. • Often referred to as minisatellites; abundance of each specific sequence at a given  location is variable – creates localized regions of 1kbp – 20kbp • Microsatellite markers: di-, tri-, tetra-, penta- nucleotide repeats; dispersed  throughout genome, varies among individuals in the number of repeats present at  each site - Middle repetitive multiple copy genes: E.g.. Genes encoding ribosomal RNA 18, 28,  5.8S and 5SrDNA. - Interspersed Retrotransposons: Short interspersed elements (SINES) and long  interspersed elements (LINES) are dispersed throughout the genome rather than  tandemly repeated, and constitute over 1/3 of the human genome.  • SINES are <500bp and may be present in 500,000 copies (13%). • LINES are ~6kbp, present in 850,000 copies (21%). • In humans, Alu is the most prevalent family of SINES, Li family is the most  prevalent LINE. - These transposable elements are generated via an RNA intermediate and are  referred to as retrotransposons Chapter 13 The Genetic Code and Transcription - General features of the genetic code: The genetic code is written in linear form, using  the ribonucleotide bases that compose mRNA molecules as “letters”; the sequence of RNA is derived from the complementary bases in the DNA; In the mRNA, triplet  codons specify one amino acid. - In the mRNA, triplet codons specify one amino acid (with 3 exceptions UGA, UAG,  UAA) - The code contains “start” and “stop” signals, certain codons that are necessary to  initiate and to terminate translation. - The genetic code is: • Unambiguous (each triplet specifies a single aa) • Degenerate (18 aa specified by more than one triplet) • Commaless (no internal ‘punctuation’) • nonoverlapping • nearly universal - Early studies established the basic operational pattern of the code - Brenner argued that the code must be a triplet. - Acridine dyes – one insertion followed by deletion restores reading frames, as does 3  insertions or 3 deletions affect only 2 codons. - Code is not overlapping – consider GTACA where parts of the central codon were  shared by the flanking codons…. - Nirenberg and Matthaei • Used an in vitro protein synthesizing system. • Used polynucleotide phosphorylase to make synthetic mRNAs • One or more radioactively labeled amino acids. - Methods: • Homopolymers • Copolymers • Triplet binding assay • Repeating copolymers- The probability of incorporation of a specific ribonucleotide is proportional to the  availability of that ribonucleotide relative to other available ribonucleotides - Nirenberg and Matthaei added RNA homopolymers to the in vitro translation system  to decipher which amino acids were encoded by the first few codons based on which  amino acids were incorporated into the polypeptide • AAA->lysine, CCC->proline, UUU->phenylalanine - Next, RNA heteropolymers were used to decipher more codons employing the same  method. - If you know the relative proportion of each type of ribonucleoside diphosphate in the  synthetic mRNA you could predict the frequency of each of the possible triplets it  contained.  - Then, by looking at the relative proportions of each amino acid incorporated in the  polypeptide the composition of the triplets specifying the amino acids can be  predicted. - Nirenberg and Leder developed the triplet binding assay to determine other specific  codon assignments. - In this technique, ribosomes bind to a single codon of three nucleotides, and the  complementary amino acid-charged tRNA will be able to bind. - Gobind Khorana: Synthesized long RNAs with di-, tri-, and tetranucleotide repeats  and used in vitro translation to determine more codons. - The Coding Dictionary reveals several interesting patterns among the 64 codons - The genetic code is degenerate, with many amino acids specified by more than one  codon. - Only tryptophan and methionine are encoded by a single codon - The First and Second position of a set of codons that specify the same amino acid  tend to be the same. - The wobble hypothesis predicts that hydrogen bonding between the codon and  anticodon at the third position is subject to modified base-pairing rules. • There are 64 possible codons, but only ~30-40 species of tRNA found in bacteria,  ~50 in plants and animals.- Chemically similar amino acids often share the middle base in the codons specifying  them. • An ordered code buffers the potential impact of mutation on protein function - The initial amino acid incorporated into all proteins is a modified form of methionine— N-formylmethionine (fmet). - AUG is the only codon to encode for methionine. - When AUG appears internally in mRNA, an unformulated methionine is inserted into  the protein. - Three codons (UAG, UAA, and UGA) serve as termination codons and do not code  for any amino acid. - Mitochondrial DNA revealed some exceptions to the universal genetic code. - Different initiation points create overlapping genes; Genetic code is non-overlapping –  each ribonucleotide in the code for a given protein is part of only one codon (reading  frame is set). - In some viruses, overlapping genes have been identified in which initiation at  different AUG positions out of frame with one another leads to distinct polypeptides - RNA serves as the intermediate molecule between DNA and proteins - RNA is synthesized on a DNA template during transcription - RNA polymerase directs RNA synthesis - RNA polymerase directs the synthesis of RNA using a DNA template. No primer is  required to initiation, and the enzyme uses ribonucleotides instead of  deoxyribonucleotides - RNA polymerase from E. coli is a holoenzyme - has multiple subunits a2, b, b', and  sigma; Core enzyme is a2bb’sigma; Sigma plays a regulatory function in the initiation  of RNA transcription; Multiple sigma factors, which create variations of the  holoenzyme; Add NTP @ 50/sec; Sigma factor is required for initiation – template  binding; Rho is required for termination. - Initiation: Transcription begins with template binding by RNA polymerase at a  promoter; the sigma subunit is responsible for promoter recognition - Promoters: These are cis elements – that is they are sequences adjacent to the gene• E. coli promoters have two consensus sequences: TATAAT (Pribnow box)  positioned at -10 from the site of transcription initiation; TTGACA positioned at –35 - Degree of binding depends on the strength of the promoter - Sigma factor recognizes promoters by scanning the DNA. - RNA polymerase binds. - DNA double helix is unwound. - Point at which transcription starts is the transcription start site. - Initiation is catalyzed – insertion of the first ribonucleoside triphosphate. - Primerless addition. - Synthesis in 5 -> 3 direction (antiparallel to template) - After ~8 nucleotides, sigma dissociates and chain elongation proceeds under the  core enzyme. - Termination: At the end of the gene, transcription terminates due to hairpin formation  in the RNA; In some cases, termination depends on the rho termination factor. - Differences between eukaryotic and prokaryotic transcription: • Transcription in eukaryotes occurs in the nucleus and is not coupled to translation. • Eukaryotes possess three forms of RNA polymerase, each of which transcribes  different types of genes • Eukaryotic transcription requires chromatin remodeling • In addition to promoters, enhancers also influence transcription regulation. Also  many trans-acting factors called TRANSCRIPTION FACTORS. • RNA transcripts are extensively processed - Eukaryotes have a nucleus. Transcription occurs in the nucleus, RNA exported to  cytoplasm for translation. Transcription and translation can be coupled in prokaryotes. - Eukaryotes possess three forms of RNA polymerase, each of which transcribes  different types of genes - RNP II requires cis- and trans- acting factors • 4 cis factors: - collectively termed ‘promoter’- core promoter element – where RNP II binds DNA - TATA box, position -30 (TATAA/TAAR) – like the -10 region in bacterial promotors. - Promoter elements (CAAT box, -80 GGCCAATCT – effects efficiency) • enhancer elements can be upstream, within, or downstream of the gene; can  modulate transcription from a distance • Silencers - Transcription factors: • GENERAL – absolutely required for RNP II transcription • SPECIFIC – influence the efficiency or rate of RNP II binding. • Designated TFIIA, TFIIB, TFIID etc. • The TATA box is a core promoter element that binds the TATA-binding protein  (TBP) of transcription factor TFIID and determines the start site of transcription.  Once TFIID binds, 7 others + RNA PolII bind to it to form an extensive pre-initiation  complex - Eukaryotic mRNA is transcribed as a precursor – much larger than what is actually  translated; Heterogeneous nuclear RNA (hnRNA) is post-transcriptionally processed  by the addition of a 5' cap and a poly-A tail; Introns are removed by splicing - The coding regions of eukaryotic genes are interrupted by intervening sequences -  introns - Introns (intervening sequences) are regions of the initial RNA transcript that are not  expressed in the amino acid sequence of the protein • 2 types of introns – type I and type II - Type I introns self splice  - Type II introns require a spliceosome; Type II introns can be very large (20kb) - Canonical splice sites 5’-GU (donor), AG-3’ (acceptor); splicesome is large; important  components are snRNPsChapter 14 Translation and Proteins  - Translation: the biological process of polymerizing amino acids into polypeptide  chains. - Amino acid sequence is directed by mRNA sequence (which is specified by DNA  sequence) - Components of the process: Amino acids, Messenger RNA (mRNA), Ribosomes and  ‘translation factors’, Transfer RNA (tRNA) and enzymes required to charge them (add  amino acid) - Francis Crick postulated the existence of an adapter molecule; this molecule (tRNA)  is an adapts specific triplet codons in mRNA to their corresponding amino acids; tRNA  is an RNA molecule that has a corresponding anti-codon triplet. - Ribosomes are large protein and RNA structures (2 subunits) that facilitate  recognition of specific codons (via hydrogen bonding) and promotes amino acid  polymerization - The rRNAs provide for important catalytic functions associated with translation. - Bacterial cell contains ~ 10,000, many more in eukaryotic cells. - rRNA genes highly redundant; E. coli has 7 copies of a sequence that produces a  30S transcript that is processed into 23S, 16S and 5S components; Hundreds of  copies of a 45S sequence in eukaryotes that is processed into 28S, 18S and 5.8S  components; 45S sequence present in clusters in mammalian genomes; 5S rRNA is  located separately. - RNA components perform the catalytic functions, proteins probably play support roles  such as promoting the binding of the various factors and may serve to fine tune the  process. - tRNA structure: Small 75-90nt; transcribed as larger precursors and cleaved into  mature tRNA molecules; several ‘unique’ nucleotides present – result from post  transcriptional modification (enzymatic); several regions of complimentary nucleotide  stretches in tRNA primary sequence yield ‘step loops’ and influence structure - Charging tRNA: tRNA charged with Aminoacyl tRNA synthetase; ~31 tRNA (out of a  possible 61 – wobble) 20 different tRNA Aminoacyl tRNA synthetases; Charging process requires activating amino acid by reaction with ATP to create an aminoacyladenylic acid ( 5’ phosphate and  corboxyl group of amino acid amino acid activation reaction occurs in association with the synthase enzyme Complex reacts with a specific  tRNA Amino acid is transferred to the appropriate tRNA – covalently  bonded to the Adenine residue at the 3’ end Aminoacyl tRNA synthases are highly specific – recognizing only One a.a. and the tRNAs corresponding to that amino acid - Translation: 3 steps: Initiation, Elongation,  Termination - Initiation (requires large and small subunit of ribosomes, GTP, charged initiator  tRNA, Mg2+ and initiation factors IF1-3); translation starts with AUG (fmet) • In bacteria, the AUG start codon is preceded by a Shine-Dalgarno sequence,  which base-pairs with a region of the 16S rRNA of the 30S small subunit; Shine Dalgarno is all purines, 6nt long (AGGAGG • Ribosomes are dissociated into large and small subunits unless active in  translation. • Small subunit binds several IFs (IFs enhance binding affinity of the various  translational components) this in turn binds mRNA. • Another initiation factor enhances binding of charged formylmethionyl tRNA to the  small subunit (in response to the presence of AUG codon). • This is the intiation complex – combines with large subunit upon hydrolysis of one  GTP.  - Elongation• Subunit assembly forms 2 tRNA binding sites. • P (peptidyl) and A (aminoacyl) site. • Peptide bond formation transfers nascent polypeptide chain to tRNA in aminoacyl  site (hydrolysis of bond between amino acid and tRNA in p site). • Uncharged amino acid exits from the exit site, tRNA bound to peptide chain moves  to the P site. - Termination is signaled by a stop codon (UAG, UAA, UGA) in the A site - GTP-dependent release factors cleave the polypeptide chain from the tRNA and  release it from the translation complex  - Polysomes (or polyribosomes) are mRNAs with several ribosomes translating at  once. - In eukaryotes:  • the ribosomes are larger than in bacteria • transcription and translation are spatially and temporally separated • Presence of 7-methylguanosine essential, 3’ poly-A required • Ribosomes scan for the initiator tRNA that is in the proper context, as identified by  the Kozak sequence (A/GNNAUGG) • Does not require formylmetionine – uses a unique tRNA tRNAimet • requires more factors for initiation, elongation, and termination. • Ribosomes are not free-floating as in bacteria but instead are associated with the  endoplasmic reticulum. - Beadle and Tatum demonstrate a link between gene and protein well in advance of  the discovery of DNA structure, transcription or translation • Through induction of metabolic mutants in Neurospora, and analysis of these to  define hierarchical metabolic pathways. • Neurospora can synthesis it’s biochemical requirements. • Used X-rays to induce mutants that could grow on complete medium but failed to  grow on minimal medium unless supplemented.• Ultimately identified several mutants that were unable to synthesis a single  molecule (amino acid, purine, pyrimidine etc.) • Convinced Beadle that genetics and biochemistry are linked: - mutations caused the loss of an enzymatic ability - Seemed likely that a mutation could be found for every enzymatic reaction. - One gene – one enzyme: Srb and Horowitz: pathway leading to synthesis of  arginine; 7 mutants (arg-) • Tested each mutant for the ability re-establish growth if either citrulline or ornithine  were present (2 molecules very similar to arginine) • arg 4-7 – grew if supplied with citrulline, or ornithine or arginine. • arg2 and arg 3 grew if supplied with citrulline or arginine • arg 1 grew only if supplemented with arginine. • LOGIC: if arg4-7 can grow regardless of which of the 3 molecules are added, the  mutation must block metabolism prior to involvement of ornithine, citrulline or  arginine. - Not all proteins are enzymes and some proteins have more than one subunit. - Because of this, the one-gene:one-enzyme hypothesis was modified to one gene:one-protein and then to one-gene:one-polypeptide chain. - Hemoglobin: not an enzyme – but a clear example of the 1 gene – 1 protein. - Sickle-cell anemia is a recessive genetic disease in which afflicted individuals are  homozygous for the HbS hemoglobin allele; Heterozygotes are carriers of the affected  gene but are largely unaffected.  - Pauling noted that a chemical difference exists between normal and sickle-cell  hemoglobin; fingerprinting demonstrated that the HbS and HbA hemoglobins differ by  a single peptide fragment; establishes that a single gene provides the genetic  information for a single polypeptide chain; demonstrates that a single amino acid  change can affect phenotype; established the concept of ‘inherited disease’ - The nucleotide sequence of a gene and the amino acid sequence of the  corresponding protein exhibit colinearity • In colinearity, the order of nucleotides in a gene correlates directly with the order  of amino acids in the corresponding polypeptide• Yanofsky fine structure mapped mutation in trpA and determined where the amino  acid substitution occurred - Variation in protein structure provides the basis of biological discovery; following  translation, polypeptides fold up and assume higher order structures (proteins), and  they may interact with other polypeptides - Amino acids all have: a carboxyl group, an amino group, an R (radical) group bound  to a central carbon atom  • The R group of an amino acid confers specific chemical properties  - Peptide bond: Covalent bond between the carboxyl group of one amino acid and the  amino group of another. Each polypeptide has an amino terminus and a carboxyl terminus  - Protein Structure: there are four levels  of protein structure:  • Primary: linear sequence of amino acids) • Secondary: alpha helix or beta pleated sheet - Tertiary: 3 important stabilizing  structures: • Covalent disulfide bonds between  close cystine residues -CH2-S-S CH2- • Hydrophilic R groups on surface • Hydrophobic R groups in interior • Ionic bonds (opposite charges) • van der Waals interaction: close polar and non-polar bonds transient fluctuations in  one induce something in the other –cause things to stick together - Quaternary: multi-subunits; subunits have conformations that facilitate their fitting  together - Post-translational modifications: • N-terminus amino acid removed or modified.• Removal of signal sequences • Individual amino acids may be modified – ie. phosphate addition • Carbohydrate addition • Polypeptide chains may be trimmed: Poly-peptide cleavage to produce an active  form. • Polypeptide chains may be complexed with metals. - Roles of Proteins: • Proteins play diverse roles in the body. • Hemoglobin binds to and transports oxygen, which is essential for cellular  metabolism. • Collagen and keratin are structural proteins. • Actin and myosin are contractile proteins found in abundance in muscle tissue. • Other examples are: - the immunoglobulins, which function in the immune system of vertebrates - transport proteins, involved in movement of molecules across membranes - some of the hormones and their receptors, which regulate various types of  chemical activity - histones, which bind to DNA in eukaryotic organisms - Enzymes, the largest group of proteins, are involved in biological catalysis, a  process whereby the energy of activation for a given reaction is lowered Chapter 15 Gene Mutation and DNA Repair - The ability of DNA to store, replicate, transfer and decode information is central to  genetic function. - Mutations, caused by mistakes in information storage or transfer is equally important  for generating diversity; allows adaptation to new environments, speciation and  evolution.- Mutations are alterations in DNA sequence; a mutation may change any part of a  DNA molecule. It may comprise a single base pair substitution, a deletion or insertion  of one or more base pairs or a major alteration in chromosome structure. - Mutations can occur in somatic cells or in the germline; transmission of genetic  diversity, evolution, genetic diseases vs. locally altered cell function - Spontaneous mutations happen naturally and randomly and are usually linked to  normal biological or chemical processes in the organism; rates of spontaneous  mutations (they are generally low) vary among organisms, and even among loci in  different organisms - Induced mutations result from the influence of an extraneous factor, either natural  or artificial. - Salvador Luria and Max Delbruck asked this question: Can organisms some how  ‘select’ or ‘direct’ the mutation of genes in order to adapt to a particular environmental  pressure? • Looked at phage resistance in E. coli. • If there is adaptive mutation, then E. coli may mutate to be phage resistant if  challenged with phage. - Adaptive mutation predictions: Small but constant probability that any given bacteria  will acquire phage resistance, so the number of resistant cells should depend only on  the number of bacteria and phage added to each plate; So, if bacteria, phage and  incubation time is constant, there should be very little fluctuation between the number  of resistant cells from plate to plate and from experiment to experiment - Spontaneous mutation predictions: If resistance is a result of randomly acquired  mutations – requiring no ‘stimulus’ from being incubated with bacteriophage –  resistance mutations will occur at a low rate during the incubation in liquid medium  prior to plating; Thus, the number of resistant bacteria seen will depend on when  during incubation the spontaneous mutation occurred; The spontaneous mutation  hypothesis predicts that the number of resistant cells will fluctuate significantly from  experiment to experiment, and from tube to tube. - Mutations are classified based on location: • Somatic mutations occur in any cell except germ cells and are not heritable.  • Germ-line mutations occur in gametes and are inherited. • Autosomal mutations occur within genes located on the autosomes, whereas  X-linked and Y-linked mutations occur within genes located on the X or Y  chromosome, respectively - Mutations can be classified based on type of molecular change: • Point mutations are base substitutions in which one base pair is altered.  • Frameshift mutations result from insertions or deletions (INDELs) of a base pair  • Missense mutations change a codon resulting in an altered amino acid within a  protein-coding portion of a gene.  • A nonsense mutation changes a codon into a stop codon and results in  premature termination of translation.  • A silent mutation alters a codon but does not result in a change in the amino acid  at that position of the protein. - If a pyrimidine replaces a pyrimidine or a purine replaces a purine, a transition has  occurred.  - If a purine and a pyrimidine are interchanged, a transversion has occurred.  - A frameshift mutation occurs when any number of bases are added or deleted,  except multiples of three, which would reestablish the initial frame of reading - Mutations can be classified according to their phenotypic effects as loss-of-function;  gain-of-function; morphological; nutritional (biochemical); behavioral; regulatory - Lethal mutations interrupt an essential process and result in death - The expression of conditional mutations depends on the environment in which the  organism finds itself; a good example is a temperature-sensitive mutation. - Neutral mutations, the vast majority of all mutations, occur in the large portions of  the genome that do not contain genes and therefore have no effect on gene products - How do spontaneous mutations arise? DNA replication errors; despite proof-reading  sometimes, DNA pol incorporates the wrong base; point mutations caused by  mispairing – sometimes caused by tautomeric shifts - Slippage during replication can lead to small insertions or deletions; template strand  may loop, or if DNA pol slips or stutters during replication.- Tautomeric Shifts: purines and pyrimidines can exist in alternate chemical forms –  differing by only a single proton shift. - DNA base damage by depurination (loss of a base) and deamination (conversion  of an amino group to a keto group) is the most common cause of spontaneous  mutation  - DNA may suffer oxidative damage by the by-products of normal cellular processes;  reactive oxygen species generated during normal aerobic respiration (superoxides  O2.- ; hydroxyl radicals (.OH) and hydrogen peroxide H2O2 - Integrations of transposons into new genomic locations (near or within genes) can  act as naturally occurring mutagens. - Induced mutations arise from DNA damage caused by chemicals and radiation • Mutagens are natural (environment) or artificial (man-made) agents that induce  mutations. • Base analogs can substitute for purines or pyrimidines during nucleic acid  replication. • Alkylating agents donate an alkyl group to amino or keto groups in nucleotides to  alter base-pairing affinity. • Acridine dyes cause frameshift mutations by intercalating between purines and  pyrimidines - UV radiation creates pyrimidine dimers that distort the DNA conformation in such a  way that errors tend to be introduced during DNA replication - Ionizing radiation in the form of X rays, gamma rays, and cosmic rays are  mutagenic (may create free radicals by ejecting electrons from the atoms of stable  molecules. - Assessing mutagenicity: the Ames test uses any of a dozen strains of Salmonella  typhimurium selected for their increased sensitivity to mutagens and their ability to  reveal the presence of specific types of mutations (each strain contains a mutation in  one of the genes of the histidine operon); assay measures the frequency of  revertants. - DNA repair systems: • Proofreading: DNA Pol III incorporates the wrong base ~ 10-5. 3->5 exonuclease  activity results in 100 fold increase in fidelity.• Mismatch repair can catch some mistakes that escape proofreading (small  insertions/deletions or bae-base mismatches that were not detected) . • How does the repair system recognize which strand is correct? - Methylation tags via adenine methylase: 5’-GATC-3’ ; 3’-CTAG-5’ - Postreplication repair responds after damaged DNA has escaped repair and failed  to be completely replicated; requires homologous recombination mediated by the  RecA protein.  - SOS repair system: activated by the presence of a large number of mismatches and  gaps (induce expression of ~20 genes involved in repair); The SOS repair system allows DNA synthesis to become error-prone; although SOS repair is itself mutagenic,  it may allow the cell to survive DNA damage that might otherwise kill it - Photoreactivation repair removes thymine dimers caused by UV light (requires  absorption of a photon of blue light); the process depends on the activity of a protein  called the photoreactivation enzyme (PRE) - Base and nucleotide excision repair: light independent repair • Involves 3 steps (cut and paste system): Remove mutation with a nuclease; Gap  filling with DNA Pol; Ligation - Two types of excision repair: base excision repair (BER) and nucleotide excision  repair. • Base excision repair (BER) involves: recognition of the erroneous base by DNA  glycosylase; cutting of the DNA backbone by AP endonuclease • Nucleotide excision repair (NER) repairs bulky lesions and involves the uvr genes  (A, B and C).  - Previous repair pathways deal with damage or errors within one strand of the DNA  helix. - DNA double-strand break (DSB) repair is activated when both DNA strands are  cleaved; It is responsible for reannealing the two strands. • Two pathways: - Homologous recombinational repair fixes a double-strand DNA break by  digesting back the 5' ends of the broken helix to leave overhanging 3' ends that interact with a region of an undamaged sister chromatid to allow DNA  polymerase to copy the undamaged DNA sequence into the damaged strand - Nonhomologous end joining repairs double-stranded breaks but does not  require a homologous region of DNA during repair; activated in G1 prior to  replication; involves a complex of 3 proteins that bind to the free ends of broken  DNA, trim the ends and ligate them back together; this is an error prone system; if more than one chromosome suffers a double strand break they could be joined  together Chapter 16 Prokaryotic Gene Regulation - ~4000 proteins in E. coli, some present in 5-10 copies/cell, some (ribosomal proteins)  ~10,000 copies - Most prokaryotic gene products are present at a basal level but the concentrations of  these can increase massively when they are required. - Fundamental regulatory mechanisms must exist to control expression. - Bacteria respond to changes in their environment – turning genes on or off  depending on the cells metabolic need for the respective gene products. - Genes expressed continuously are constitutive - Genes that are turned on (assayed by an increase in enzyme) in response to the  presence of a chemical substrate are inducible; examples include lactose and the lac  operon - There exists a contrasting system: the presence of a specific molecule inhibits gene  expression – repressible; such molecules are usually the end products of anabolic  biosynthetic pathways; an example is tryptophan – plays a role in repressing the  synthesis of mRNA required to produce tryptophan-synthesizing enzymes - Regulation, whether of the inducible or repressible type, may be under negative or  positive control - Negative control: expression occurs unless it is shut down by some sort of regulator  molecule.- Positive control: transcription occurs only if a regulator molecule directly stimulates  RNA production. - In theory, either type of control or a combination of the two can govern inducible or  repressible systems - Lactose metabolism: enzymes responsible for lactose metabolism are inducible –  lactose is the inducer; In the presence of lactose the concentrations of the enzymes  required for its metabolism increase rapidly from a few molecules to thousands/cell - Bacterial genes that code for related functions are often organized into clusters. - Transcription of these is often under the coordinated control of a single regulatory  region. - These regulatory regions are usually upstream (5’) of the gene cluster it controls.  These are on the same strand as the gene cluster and said to be cis - Cis acting elements bind molecules that control transcription of the cluster – these  are trans factors. - Binding of a trans-acting element at a cis-acting site can regulate the gene cluster  either negatively (turn off transcription) or positively (turn on transcription) - The lac operon has three structural genes, lacZ, lacY, and lacA, with an upstream  regulatory region consisting of an operator and a promoter - The lacZ gene encodes -galactosidase, an enzyme that converts the disaccharide  lactose to the monosaccharides glucose and galactose - The lacY gene specifies the primary structure of permease, an enzyme that  facilitates the entry of lactose into the bacterial cell - The lacA gene codes for the enzyme transacetylase, which may be involved in the  removal of toxic by-products of lactose digestion from the cell - Genetics of the lac operon: structural genes of the lac operon are transcribed as a  polycistronic mRNA • Constitutive mutants produce enzymes involved in lactose metabolism regardless  of the presence or absence of lactose. • lacI- mutant mapped to a region close to, but distinct from the structural genes lac  Z, lacY and LacA -> led to the discovery of the lacI gene which is the lac repressor.• A second set of constitutive mutants defined a locus adjacent to the structural  genes – called the operator region.  - Jacob and Monod – proposed the operon model in which a group of genes is  regulated and expressed as a unit; Repressor normally binds operator region –  inhibits the action of RNA polymerase; Lactose present, lactose binds to the  repressor, which alters the binding site of the repressor; Because transcription occurs  only when the repressor FAILS TO BIND the operator region regulation is said to be  under negative control - Lactose induction summary • Thus the operon model invokes a series of molecular interactions between  proteins, inducers, and DNA for efficient regulation of structural gene expression • No lactose, enzymes not needed, expression of genes encoding enzymes  repressed • Lactose present; indirectly induces activation of genes by binding to repressor • All lactose metabolized, none available to bind to repressor, transcription  repressed - Proof of the model based on genetic analysis: • 4 testable predictions from the model: • I gene produces a diffusible product (trans-acting) • O region is involved in regulation, but does not produce a product. (cis acting). • O region must be adjacent to the structural genes in order to regulate their  transcription. • There should be mutant alleles of I that always bind to the operator (do not bind  lactose) • Analysis of lac expression in the absence or presence of lactose in partial diploid  merozygotes (recall chapter 6) was used to prove the operon model for the lac operon; for example, can use an F’ plasmid to introduce an I+ gene to a host that  was I- - Catabolite-Activating protein (CAP) exerts positive control over the lac- operon - The catabolite-activating protein (CAP) is involved in repressing expression of the  lac operon when glucose is present; this inhibition is called catabolite repression.- In the absence of glucose and the presence of lactose, CAP exerts positive control by  binding the CAP-binding site and facilitating RNA polymerase binding at the promoter - For maximal expression, the repressor must not be bound at the operator and CAP  must be bound at the CAP-binding site - How is the presence of glucose inhibit CAP binding? Cyclic adenosine  monophosphate (cAMP) is required for CAP binding; Glucose represses expression  of adenylyl cyclase, the enzyme that catalyzes the production of cAMP , and thus  prevents CAP from binding when glucose is present - The Tryptophan (trp) operon in E. coli Is a repressible gene system; the enzymes for  tryptophan production form an operon; in the presence of tryptophan, the operon is  repressed; contrast this to the lac operon which is an inducible gene system: because  presence of the substrate induces transcription) - When Trp is present, tryptophan synthase and associated proteins are not produced;  tryptophan functions as a corepressor, which is required for the repressor to bind to  the operator - Genetic evidence for the Trp operon model: 2 classes of mutants. • Class 1: TrpR- maps away from the structural genes. No repression regardless of  trp presence • Class 2: maps adjacent to the structural genes; analogous to the operator of the  lac operon – sequence fails to interact with repressor. - Molecular analysis provides evidence for another layer of regulation; entire Trp  operon now well defined; 5 structural genes are transcribed as a polycistronic  message. • trpP represents binding site for RNA pol • trpO is the operator region (binds the repressor) • In the absence of binding, transcription is initiated within the trpP-trpO region and  proceeds along a leader sequence 162 nt prior to the first structural gene. - In the presence of tryptophan, even when the trp operon is effectively repressed,  Initiation of transcription still occurs –so repressor binding must not be strictly  repressive; So, there must be subsequent mechanism by which tryptophan must  inhibit the transcription of the rest of the message; Found that in the presence of  trypothan, transcription was terminated at a point 140bp along the transcript; This type of repression, in which transcription of the operon is greatly reduced, but not  eliminated entirely, is called attenuation - In the absence of tryptophan, transcription is not terminated in the leader region and  proceeds through the entire operon - The leader region can form two different conformations, depending on the presence  or absence of tryptophan; the leader region contains two tryptophan codons. - In the presence of tryptophan, the hairpin structures formed act as a transcriptional  terminator - The antiterminator hairpin structure forms in the absence of tryptophan because  the ribosome stalls at these codons because there is not adequate charged tRNAtrp.  - In the presence of tryptophan, the ribosome proceeds through this sequence and the  terminator hairpin can form - The attenuation mechanism is common to several operons for enzymes responsible  for synthesis of other amino acids; these include threonine, histidine, leucine, and  phenylalanine. - Riboswitches: numerous cases of gene regulation depend on alternative forms of  mRNA secondary structure and involve riboswitches; riboswitches are mRNA  sequences present upstream from the coding sequence – in the 5’ UTR; mRNA  responds to the environment of the cell and regulates its own expression - All riboswitches posses a metabolite-sensing RNA sequence that allows transcription  of that RNA to either proceed or not proceed - Two important domains within a riboswitch are the aptamer, which binds to the  ligand, and expression platform, which is capable of forming the terminator structure - The arabinose (ara) operon is subject to both positive and negative regulation by  the AraC protein • AraC interacts with two regulatory regions, araI and araO2 • AraC binds to araI in the presence of arabinose and CAP-cAMP to induce  expression • In the absence of arabinose and CAP-cAMP, AraC binds to both araI and araO2 to  form a loop that causes repressionChapter 17 Eukaryotic Gene Regulation - Gene regulation in eukaryotes is more complex than it is in prokaryotes because of: • the larger amount of DNA • Complexed with histones • larger number of chromosomes • spatial separation of transcription and translation • mRNA processing • RNA stability • cellular differentiation in eukaryotes - Programmed DNA rearrangements expression of a small number of genes - Eukaryotic gene expression is influenced by chromatin modifications  - Transcription initiation is regulated at specific Cis-acting sites - Transcription initiation is regulated by transcription factors that bind to Cis-acting  sites - Activators and Repressors interact with general transcription factors at the promoter - Gene expression control through RNA silencing  - Chromosome Territories: chromosomes occupy a discrete territory in the nucleus and  stay separate from other chromosomes - During interphase chromosomes are unwound and can not be seen by microscopy.  Chromosome painting has demonstrated that each chromosome occupies a discrete  domain in the nucleus: chromosome territory - Channels between chromosomes are called interchromosomal domains - Chromosome structure is continuously rearranged so that transcriptionally active  genes are cycled to the edges of chromosome territories - Transcription factories are nuclear sites that contain most of the active RNA  polymerase and transcription regulatory molecules- Chromatin remodeling: The ability of a cell to alter the associated of DNA with other  chromatin components is crucial to allow regulatory elements to access DNA. - Chromatin can be remodeled in three ways: Changes to nucleosomes by altering  nucleosome components; Reposition or removal of a nucleosome on a gene region;  Changes to the DNA itself through covalent modifications - Chromatin remodeling must occur to allow the DNA to be accessed by DNA binding  proteins - Most histones contain normal histones H2A and H3; variant histones (H2A.Z and  H3.3) can facilitate gene transcription - Histone tails are important for histone modifications such as acetylation,  methylation, and phosphorylation - Histone acetylation and methylation are associated with increase in gene activity - Phosphorylation is seen at characteristic times throughout the cell cycle - Most nucleosomes contain H2A, but promoter regions of transcriptionally active  genes are often flanked by nucleosomes containing variant histones (H2A.Z) - Histone modification: Histone acetyltransferases (HATs) add acetate groups to  certain a.a.s of the histone – reduces attraction between basic histones and acidic  DNA; HATs are recruited by specific transcription factors. Loosening of histones from  DNA facilitates further remodeling catalyzed by ATP-dependant remodeling  complexes - DNA methylation is associated with decreased gene expression; Methylation occurs  most often on the cytosine of CG doubled in DNA; Methylation can repress  transcription by binding to transcription factors of DNA - Transcription initiation is regulated by Cis-acting sites; eukaryotic transcription  regulation requires the binding of regulatory factors (trans) to specific DNA sequences  located in and around genes; 3 classes: Promoters, enhancers, silencers - Promoters: nucleotide sequences that serve as recognition sites for the  transcriptional machinery • Located adjacent to the genes they regulate. • Specify site(s) of transciption, and the direction  • May be long (~several 100bp)- Cis-acting sites: Promoters • 2 subcategories: - Core promoter: determines the accurate initiation of transcription by RNA Pol-II - Proximal Promoter Elements: these modulate the efficiency of basal levels of  transcription. • Great diversity exits in promoters in terms of structure and function (Figure 17.3) - Focused promoters: specific transcription start site; highly-regulated genes • Dispersed promoters: Several start sites; associated with constitutive expression;  Associated with CpG islands - Promoters are made up of one or more core elements that bind to specific initiation  proteins • Initiator (Inr): YYANA/TYY, +2 to -4, includes TSS,  • TATA box: TATAA/TAAR, -30 • TFIIB recognition element (BRE): immediately up or down from TATA. • downstream promoter element (DPE) +18-27 • motif ten element (MTE) +28-32 • Many promoters contain proximal promoter elements located upstream of the TATA  and BRE motifs, enhancing levels of basal transcription - CAAT: (CAAT or CCAAT), -70-80 - GC boxe(s): (GGGCGG), -110  - Basal (general) transcription factors are required for the binding of RNA  polymerase II to the promoter Pre-initiation complex (PIC) - TFIID, the first general transcription factor to bind the promoter, binds to the TATA  box through the TATA binding protein (TBP) - Transcription initiation is regulated by Trans factors - Transcription regulatory proteins, transcription factors, target cis-acting sites of  genes regulating expression • Activators increase transcription initiation• Repressors decrease transcription initiation - The effects regulatory proteins can be finely turned to the appropriate cell type in  response to environmental cues or during development - Transcription factors: proteins that serve as transcription factors have two functional  domains (clusters of amino acids with a specific function): • A DNA-binding domain: Binds to specific DNA sequences in the cis-acting  regulatory site • A trans-activating domain: activates or represses transcription by binding to other  transcription factors or RNA polymerase - Characteristic domains of DNA-binding proteins include • helix-turn-helix (HTH) • zinc finger • basic leucine zipper (bZIP) - Transcription initiation is regulated by Trans factors - The human metallothionein IIA gene (hMTIIA) provides an example of how a gene  can be transcriptionally regulated through the interplay of multiple promoter and  enhancer elements and the transcription factors that bind to them  - Enhancers (cis-acting) are modular and contain several short DNA sequences  increasing transcription rates - 3 features of enhancers: • Located on either side of gene, some distance from gene, or even within the gene • Can be inverted without significant effect • General – act on genes ‘close’ to them - Silencers are cis-acting elements that repress the level of transcription initiation - Activators and repressors interact with general transcription factors at the promoter - After chromatin has been remodeled, transcription factors can bind their Cis factors  to bring about positive and negative effects on the transcription initiation. - Activators and repressors may increase or decrease the rate of transcription initiation  in several ways:• Activators/repressors may bind to chromatin near the promoter and open/close  regions of promoter • Activators/repressors may bind directly to transcription factors to enhance or inhibit  initiation • Activators bind to enhancers and form the enhanceosome, which interacts with  the transcription complex • Repressor proteins bind at silencer DNA elements to repress transcription - Gene regulation in a model organism: transcription of the GAL genes of yeast - The GAL system in yeast is made up of four structural genes and three regulatory  genes • Structural:GAL1, GAL10, GAL12, GAL7 • Regulatory: GAL4, GAL80, GAL3 - The products of the structural genes transport galactose into the cell for metabolism - The products of the regulatory genes positively/negatively control transcription of  structural genes - The GAL genes are inducible (transcribed) by the presence of galactose, but only if  the concentration of glucose is low - This indicates that the GAL genes are also subject to catabolite repression - The GAL1 and GAL10 genes are controlled by a central control region, UASG, that  contains four binding sites for the Gal4 protein (Gal4p)  • UAS are functionally similar to enhancers in eukaryotes - The chromatin structure of a UAS is constitutively open, or DNase hypersensitive,  meaning that it is free of nucleosomes - Post-transcriptional Gene Regulation: although transcriptional control is perhaps the  major type of regulation in eukaryotes, posttranscriptional regulation plays an  equal if not more significant role; this includes:  • removal of introns and splicing together of exons • addition of a cap and poly-A tail • translation• stability - mRNA Stability: steady-state level of an mRNA determined by a combination of  transcription rate and degradation rate. - mRNA lifetime is described in terms of it’s half-life (t1/2). - The abundance of some proteins involved in regulating transcription, cell growth and  differentiation is determined more by controlling the rate of degradation of the mRNA  for those proteins - Some mRNAs are degraded within minutes of their synthesis – others are around for  years - Three general paths for mRNA degradation: • An mRNA may be targeted by enzymes that shorted it’s polyA tail - polyA tail ~200nt – binds polyA binding proteins. If the tail is reduced to <30, the  mRNA becomes unstable and is targeted by exonucleases. • mRNA may be de-capped • mRNA may be cleaved by an endonuclease creating unprotected ends that are  subjected to exonucleases; e.g. NMD and RNA interference - Alternative splicing can generate different forms of mRNA from identical pre mRNA, giving rise to a number of proteins from one gene - As a result, the number of proteins that a cell can make (its proteome) is not directly  related to the number of genes in the genome - Approximately 90% of human genes undergo alternative splicing - Humans produce several hundred thousand different proteins from approximately  25,000 genes in the haploid genome - Mutations that affect regulation of splicing (spliceopathies) contribute to several  genetic disorders:Myotonic dystrophy, fragile-X, Huntington - Does the Drosophila nervous system really need all these alternatives? • Evidence shows that each neuron expresses a different subset of Dscam protein  isoforms. • In vivo studies show that each Dscam protein isoform can bind to the same Dscam  protein isoform, but not others.• Perhaps the diversity of Dscam protein isoforms in neurons provides a molecular  identity tag for each neuron, which helps to guide it to the corect target, and prevent  tangling of unrelated neurons. - Spliceopathies: mutations that affect regulation of splicing (spliceopathies)  contribute to several genetic disorders • Myotonic dystrophy (DM), fragile-X, Huntington • DM most common form of adult muscular dystrophy (1/8000) • Autosomal dominant, 2 forms DM1, DM2 • DM1 is caused by the expansion of a trinucleotide repeat (CTG). 5-35 copies are  normal, 150-2000 copies result in DM • DM2 is caused by expansion of CCTG within the first intron of the ZNF9 gene. - A.S. and Sex Determination in Drosophila: Recall, sex in Drosophila is determined by  the ratio of X chromosomes to sets of autosomes. So 1X:2A is male regardless of the  presence of a Y; Chromosomal ratios are interpreted by genes that initiate a cascade  of splicing events - These result in production of male or female somatic ceels, and the corresponding  male or female phenotyoes. - 3 major genes in this pathway are: • Sex lethal (Sxl): Sxl encodes an RNA binding protein; activation by TFs on the X;  activation is concentration dependent; Presence of SXL protein begins cascades of  female specific splicing events – overrides default male splicing patterns • transformer (tra) • doublesex (dsx) - In some cases translation of an mRNA can be regulated by the need for the protein  product. • e.g. the control of ferritin and transferrin receptor mRNA translation. • Soluble iron required for many enzymes, but high levels are toxic. • Iron usually bound to a protein called transferrin which is involved in moving it into  cells. • In cytoplasm, iron is bound to ferritin to ‘inactivate it’.- Levels of ferritin must be regulated in response to iron levels. - Levels of transferrin receptor must be regulated to provide enough intracellular iron. - RNA silencing controls gene expression in several ways: • Short RNA molecules regulate gene expression in the cytoplasm of plants,  animals, and fungi by repressing translation and triggering mRNA degradation • This form of sequence-specific posttranscriptional regulation is known as RNA  interference (RNAi) • Together, these phenomena are known as RNA-induced gene silencing • Gene silencing by RNA interference (RNAi) uses a protein called Dicer to cleave  double-stranded RNA molecules into small interfering RNAs (siRNAs) that bind  to the RNA-induced silencing complex (RISC complex) for unwinding. • The single-stranded RNAs target mRNAs with complementary sequences to mark  them for degradation  - Programmed DNA Rearrangements: genomic DNA is generally ‘stable’, but some  developmental gene regulation requires precise, programmed, gene rearrangements;  this is typified by the regulation of immune-system genes expression; rearrangements  result in loss and rearrangement of gene segments within particular somatic cells - Immune system components: Components fall into 2 major classes: • Humoral immunity involves the production of proteins called immunoglobulins or  antibodies that bind directly to antigens, Cellular identity involves the recognition of  antigens that are present on the surface of cells, by T lymphocytes or T cells;  Consider antibodies that are synthesized by B lymphocytes - Immunoglobulin structure: Immunoglobulins consist of four polypeptide chains: • 2 light (L) chains: 2 types of light chains in humans (kappa, Ch2 and lambda,  CH22). • 2 heavy (H) chains: 1 type of heavy chain – Ch14 • Each contain constant and variable regions  - Immunoglobulins that contain either kappa or lambda light chains can exist in 5  classes (IgM, D, E, G A) – each class performs different functions. • IgM present on surface of immature B cells• IgG and IgA are secreted by mature B cells - In the presence of an antigen, the B cells that recognize it divide and develop; Immunoglobulins are first expressed in IgM, then one of the other forms after B cell  maturation and development. - Specificity in dictated by the Variable regions, immunoglobulin class determined by  the constant regions - Hundreds of millions of varieties of antibodies are created by somatic recombination  of the variable, joining, and constant regions; Somatic recombination is mediated by  the RAG1 and RAG2 proteins • Consider the kappa light chains as an example: - The mature kappa light chain is made up of three regions: Variable, Joining,  Constant - The kappa protein expressed is not encoded by a single germ line gene, but by  a gene that is assembled in the somatic B cell from multiple gene regions on  CH2. - Heavy chains: rearrangements are more complex; 4 segments (V, D, J and a C  region); 300 V regions, 20 D, 6 J and 9 C - Immunoglobulin and TCR diversity is mediated by programmed DNA  rearrangements.  - The following features contribute to the diversity: • Multiple different V regions. • Multiple different D and J regions. • Imprecise recombination that leads to nucleotide variability at the recombination  junctions.  • Hypermutable V regions • Class switching within the C regions of the heavy chains • Various combination of L and H chains to form a mature immunoglobulin protein

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