Transcription Translation and Replication Notes

NOTES NOTES TRANSCRIPTION, TRANSLATION, & REPLICATION DNA STRUCTURE osms.it/DNA-structure DNA (DEOXYRIBONUCLEIC ACID) ▪ Two polynucleotide chains (double helix shape) Nucleotides ▪ 5-carbon sugar, phosphate group, nitrogenous base Sugar ▪ Deoxyribose in DNA, ribose in RNA Nucleobases ▪ Purines: adenine (A), guanine (G) ▫ Pure silver: purines (pure), adenine, guanine (AG) ▪ Pyrimidines: cytosine (C), thymine (T) for DNA, uracil (U) for RNA ▫ Mnemonic: CUT the PYE MNEMONIC: CUT the PYE Pyrimidines Cytosine Uracil Thymine The PYrimidinEs Figure 42.1 Nucleotides consist of a phosphate group, 5-carbon sugar (deoxyribose for DNA) and a nitrogenous base. The base can be a purine, which has two rings (adenine, guanine), or a pyrimidine, which has one ring (cytosine, guanine). OSMOSIS.ORG 349

Nucleotide binding and bonding ▪ Nucleotides bind using sugar, phosphate groups (phosphate group on 5th carbon of sugar binds covalently to 3rd carbon of sugar) → sugar-phosphate backbone ▪ Nucleotides form hydrogen bonds with bases on opposing strand ▫ Complementary base pairing: A pairs with T/U (two hydrogen bonds), C pairs with G (three hydrogen bonds) DNA structure and packing ▪ Strands coil around each other once every 10 base pairs → major, minor grooves ▪ In order to be packed tightly, DNA wrapped around histones (positive charge attracts to negative charge of phosphate backbone) → nucleosomes ▪ Nucleosomes further packed as chromatin fibers ▫ Euchromatin: loosely packed (genes frequently used) ▫ Heterochromatin: densely packed (genes rarely used) Figure 42.2 Nucleotide binding: phosphate group on 5th carbon of sugar on one nucleotide (called 5 prime carbon) binds covalently to 3rd carbon of sugar on another nucleotide (called 3 prime carbon. This gives each DNA strand a sugar-phosphate backbone and a direction (5’ to 3’ and 3’ to 5’). Nucleotide bonding: nucleotide bases form hydrogen bonds with the complementary base on the opposing strand, A with T (U in RNA) and C with G. Figure 42.3 Major and minor grooves: larger/ smaller spaces between DNA strands where proteins can bind to regulate functions. 350 OSMOSIS.ORG Figure 42.4 DNA wraps around histone proteins to form nucleosomes, which pack tighter again to form chromatin fibers.

Chapter 42 Genetics: Transcription, Translation, & Replication DNA REPLICATION osms.it/DNA-replication ▪ Occurs in S phase of cell cycle (before cell division) ▪ 46 chromosomes duplicated → each daughter cell gets genetic material ▪ DNA replication semiconservative → each strand of double helix template PROCESS Initiation ▪ Pre-replication complex seeks origin of replication, DNA helicase splits strands → replication fork ▫ Single-stranded DNA binding proteins improve stability of lone strands ▫ DNA topoisomerase prevents overwinding of later DNA Elongation ▪ RNA primase creates multiple RNA primers → randomly bind → DNA polymerase adds complementary nucleotides in 3’, 5’ direction ▫ Forms single leading strand ▫ Forms single lagging strand by attaching (with DNA ligase) multiple Okazaki fragments DNA CLONING ▪ Technique used to duplicate segment of DNA within host organism ▪ Uses “plasmids”: genetic structures outside of chromosomes, replicate independently Process ▪ Extract desired DNA segment using specific restriction enzymes ▪ Paste segment into plasmid with DNA ligase → “recombinant DNA” ▪ Insert plasmid into host organism (e.g. E. coli), encouraging uptake with shock (e.g. heat) ▪ Identify bacteria carrying plasmid with antibiotics (plasmids given antibiotic resistance gene) ▪ Leave bacteria to replicate DNA segment, mass-manufacture protein(s) Applications ▪ Producing biopharmaceuticals (e.g. insulin), gene therapy (e.g. cystic fibrosis) Termination ▪ DNA polymerase leaves strand at telomere (TTAGGG nucleotide sequences) ▪ Hayflick limit: maximum number of times cell’s DNA can be replicated ▫ Due to repeated shortening of telomeres during termination step OSMOSIS.ORG 351

Figure 42.5 Three steps of DNA replication: initiation, elongation, and termination. DNA replication results in two sets of identical DNA, each containing one old strand and one new one. 352 OSMOSIS.ORG

Chapter 42 Genetics: Transcription, Translation, & Replication Figure 42.6 DNA cloning. Restriction enzyme (in this case, EcoRI) cleaves a known sequence surrounding a target gene and a plasmid, creating pieces with sticky ends. When DNA ligase is added, these pieces form recombinant DNA (plasmid containing target gene), as well as a gene for antibiotic resistance. A host, in this case E. coli, is combined with recombinant plasmids and subjected to a stressor so that some bacteria take up plasmid. Bacteria are allowed to replicate on plate containing antibiotic, so that only ones that have taken up plasmid can survive. These bacteria produce desired protein from target gene in plasmid. OSMOSIS.ORG 353

TRANSCRIPTION osms.it/transcription ▪ First step in creating protein from gene ▪ Gene read, copied on individual messenger RNA (mRNA) PROCESS ▪ DNA unpacked from chromatin, undergoes dehelicization ▪ Promoter region identifies starting point for transcription (e.g. TATA box) ▪ RNA polymerase shears hydrogen bonds between two strands → transcription bubble ▪ RNA polymerase follows template strand to assemble mRNA molecule (complementary to template strand) ▪ Hydrogen bonds reform on nucleotides (already transcribed) ▪ Termination sequences contains two complementary sequences → resulting mRNA binds with itself forming hairpin loop ▪ RNA polymerase detaches, DNA closes back up ▪ Polyadenylate polymerase adds 7-methyl guanosine cap to 5’, polyadenine tail to 3’ end of mRNA ▪ Spliceosomes remove introns (don’t code proteins) to leave behind exons (do code proteins) ▪ Resulting mRNA processed by ribosome to create desired protein (translation) Figure 42.7 Transcription. 1: DNA unpackaging, dehelicization; promoter region identified (TATA box); RNA polymerase shears hydrogen bonds between strands → transcription bubble. 2: RNA polymerase assembles mRNA strand complementary to template strand. Hydrogen bonds reform between DNA nucleotides already transcribed. 3: Termination sequence causes mRNA to form hairpin loop, detach. 4: Cap and tail added, introns spliced out. 354 OSMOSIS.ORG

Chapter 42 Genetics: Transcription, Translation, & Replication Figure 42.8 One strand of DNA is called the coding strand and the other is called the template strand. They have complementary nucleotide sequences. RNA polymerase builds an mRNA molecule by reading the template strand and adding complementary nucleotides. Therefore, the mRNA will have the same sequence and directionality as the coding strand, only with U instead of T. TRANSLATION osms.it/translation ▪ Second step in creating protein from gene ▪ Ribosomes assemble protein from mRNA template produced in transcription PROCESS ▪ mRNA floats out of nucleus through pore ▪ Initiation: ribosome grabs mRNA, finds start codon (e.g. AUG) ▪ Elongation: ribosome moves along mRNA, producing specific amino acid for each codon ▪ Termination: ribosome reaches stop codon, releases polypeptide (e.g. UGA) ▪ Binds to ribosome on aminoacyl/peptidyl/ exit site ▫ Aminoacyl: binds transfer RNA (tRNA) with complementary mRNA codon ▫ Peptidyl: holds tRNA with polypeptide ▫ Exit: holds tRNA after amino acid released TRANSFER RNA (tRNA) ▪ Finds, carries amino acids to ribosome ▪ Three-letter coding sequence (complementary to mRNA) Figure 42.9 Ribosome binding sites. OSMOSIS.ORG 355

Figure 42.10 One strand of DNA is called the coding strand and the other is called the template strand. They have complementary nucleotide sequences. RNA polymerase builds an mRNA molecule by reading the template strand and adding complementary nucleotides. Therefore, the mRNA will have the same sequence and directionality as the coding strand, only with U instead of T. Figure 42.11 Translation extending an existing polypeptide chain. 1: tRNA with amino acid and codon complementary to that of mRNA binds at ribosome A site. 2: Peptide bond forms between amino acid on new tRNA and tRNA in P site holding polypeptide chain, polypeptide chain is transferred to tRNA in A site. 3: Everything moves by one site. A site is now open for a new tRNA. 356 OSMOSIS.ORG

Chapter 42 Genetics: Transcription, Translation, & Replication CELL CYCLE osms.it/cell-cycle ▪ Sequence of events between formation, division of somatic cell ▪ Two phases ▫ Interphase: preparatory phase; cell performs basic functions, replicates DNA ▫ Mitosis: cellular division G0 (G-ZERO) PHASE ▪ Cells function but not dividing/preparing to divide ▪ Considered outside cell cycle INTERPHASE ▪ Terminates with G1 checkpoint ▫ Cells with damaged DNA → G0 phase/ apoptosis Synthesis (S) phase ▪ DNA replicated (identical chromatids created) Gap/Growth 2 (G2) phase ▪ Organelles duplicated ▪ Terminates with G2 checkpoint MITOSIS (M) PHASE ▪ Cell divides into two daughter cells ▪ Three subphases: G1, S, G2 phases Gap/Growth 1 (G1) phase ▪ Longest phase ▪ Cell grows while organelles function as usual Figure 42.12 Cell cycle summary. OSMOSIS.ORG 357

MITOSIS & MEIOSIS osms.it/mitosis-and-meiosis ▪ Two processes of cell division MITOSIS ▪ Division of cell into two identical daughter cells ▪ Part of cell cycle ▪ Consists of prophase, metaphase, anaphase, telophase Prophase ▪ Chromatin fibers condense ▪ Centrioles align chromosomes between centrosomes Metaphase ▪ Prometaphase: nuclear membrane, nucleolus disintegrate ▪ Metaphase: chromosomes align along metaphase plate, spindle fibers attach to kinetochores Anaphase ▪ Centrosomes pull on spindle fibers to separate chromatids Telophase ▪ New nuclear envelopes form MEIOSIS ▪ Division of cell into four haploid daughter cells ▪ Consists of ▫ Meiosis I: prophase I, metaphase I, anaphase I, telophase I ▫ Meiosis II: prophase II, metaphase II, anaphase II, telophase II Meiosis I ▪ Prophase I ▫ Leptotene: 46 chromosomes condense, nuclear membrane disintegrates ▫ Zygotene: chromosomes find homologues, bind, forming tetrads (AKA synapsis) 358 OSMOSIS.ORG Figure 42.13 Stages of mitosis: division of one cell into two identical daughter cells.

Chapter 42 Genetics: Transcription, Translation, & Replication ▪ ▪ ▪ ▪ ▫ Pachytene: homologous chromosomes exchange genetic material (AKA crossing-over) ▫ Diplotene: homologous chromosomes uncoil, slide toward ends (AKA chiasmata) ▫ Diakinesis: terminalization completed Metaphase I ▫ Tetrads migrate to metaphase plate Anaphase I ▫ Tetrads split up ▫ Chromosomes pulled to each pole by spindle fibers ▫ Diploid cell → haploid cell Telophase I ▫ Cleavage furrow appears, cytokinesis occurs Followed by interphase without chromosome duplication in S phase Meiosis II ▪ Meiosis II progresses exactly as mitosis ▫ Two haploid cells → four haploid cells ▫ Same phase names Figure 42.15 Meiosis produces haploid daughter cells with 23 chromosomes each. Figure 42.14 Steps of meiosis I, prophase I. OSMOSIS.ORG 359

GENETIC MUTATIONS & REPAIR osms.it/DNA-mutations osms.it/DNA-damage-and-repair DNA MUTATIONS ▪ Alterations in nucleotide (A, T, G, C) sequence of ≥ one gene ▫ Affect somatic cells (AKA nonreproductive cells), gametes → germline mutations ▫ Arise spontaneously/due to mutagens SMALL-SCALE MUTATIONS ▪ Single gene ▪ Substitutions: nucleotide replaced by another ▪ May result in ▫ Silent mutation: same amino acid ▫ Missense mutation: different amino acid (e.g. sickle cell disease) ▫ Nonsense mutation: stop codon INSERTIONS & DELETIONS ▪ Nucleotide added/removed from sequence ▪ Multiples of three → nonframeshift mutation ▫ Reading frame displaced by entire codon → remaining amino acids unchanged → similar resulting protein ▪ Frameshift mutation: resulting protein abnormally long/short, most likely nonfunctional LARGE-SCALE MUTATIONS ▪ Often occur due to errors in gamete formation Abnormal number of chromosomes ▪ Aneuploidy ▫ Additional chromosomes (e.g. Down syndrome) ▫ Missing chromosomes (e.g. Turner’s syndrome) ▪ Polyploidy ▫ Increased number of chromosomes per set (e.g. triploidy) Figure 42.16 Small-scale mutations include: substitutions, deletions, and insertions. They may have a small or large effect on protein function depending on how the new nucleotide affects the translation of the codon sequence into amino acids. 360 OSMOSIS.ORG

Chapter 42 Genetics: Transcription, Translation, & Replication Structurally abnormal ▪ Movement of sections of chromosomes ▪ Deletion: part of chromosome goes missing (e.g. cri du chat syndrome) ▪ Duplication: part of chromosome duplicated ▪ Inversion: part of chromosome breaks off, reattaches ▪ Translocation: parts of two chromosomes switched Figure 42.17 Aneuploidy and polyploidy are types of large-scale mutations which result in an abnormal number of chromosomes. Figure 42.18 Illustration of types of structural abnormalities. OSMOSIS.ORG 361

DNA DAMAGE ▪ DNA damaged by endogenous, exogenous (environmental) factors ▪ If damaged DNA cannot be fixed → multiple paths ▫ Senescence: stops dividing ▫ Apoptosis: programmed cell death ▫ Uncontrolled cell division: develops into tumor ▪ If damaged DNA can be fixed → G0 phase Single strand damage ▪ Causes ▫ Endogenous (errors in DNA replication) ▫ Exogenous (harmful chemical/physical agents) ▪ Repaired with mismatch/base excision/ nucleotide excision repair ▫ Endonucleases cleave damaged segment ▫ Exonucleases remove damaged segment ▫ DNA polymerase rebuilds segment ▫ DNA ligase glues new segment Double stranded breaks ▪ May be due to ionizing radiation Repair mechanisms ▪ Non-homologous end joining ▫ DNA protein kinase binds to each end of the broken DNA → artemis cuts off rough ends → ends are rejoined with DNA ligase ▪ Homologous end joining ▫ MRN protein complex binds to each end and removes affected nucleotides → DNA polymerase copies genetic information from sister chromatid Figure 42.19 Repair of a mismatched nucleotide on a newly synthesized DNA strand. 1: Endonucleases cleave either side of damaged segment; 2: Exonucleases remove damaged segment; 3: DNA polymerase rebuilds segment; 4: DNA ligase connects new segment to strand. 362 OSMOSIS.ORG

Chapter 42 Genetics: Transcription, Translation, & Replication Figure 42.20 Two repair mechanisms for double-stranded breaks: non-homologous end joining and homologous recombination. OSMOSIS.ORG 363