Ch.8 Microbial Genetics Replication and Protein Synthesis PDF

Ch.8: Microbial Genetics poly-ribosomal complex poly-ribosomal complex – SEM micrograph  Ch. 8 – Learning Outcomes: Describe the structure and replication of bacterial genomes. Explain how genes are transcribed to RNA and translated to protein...

Ch.8: Microbial Genetics poly-ribosomal complex poly-ribosomal complex – SEM micrograph  Ch. 8 – Learning Outcomes: Describe the structure and replication of bacterial genomes. Explain how genes are transcribed to RNA and translated to protein. Explain how gene expression of the lac operon is regulated. Define a mutation and their causes. Compare / contrast point mutations and insertion/deletion mutations. Describe three methods of horizontal gene transfer.  Genetic Terms: Genetics: o the study of genes, how they carry information, how information is expressed, and how genes are replicated Genome: all the genetic information in a cell Chromosomes: o organized long strands of DNA that physically carry hereditary information; each chromosome contains specific genes Gene: o segment of DNA that encodes for specific proteins, or set’s of protein’s, that code for a specific trait or characteristic Allele: o one of two or more alternative forms of a gene found at the same place on a homologous chromosome pair. Each allele represents the male or female version of the gene. Genotype: the specific allele combinations of an organism for a specific trait (what you have) Phenotype: physical expression of that trait (what you see)  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types  DNA and Proteins…. the Big Picture: o The science of heredity o Central dogma of molecular biology o Mutations o Gene expression controlled by operons steps in the central dogma of biology and how they are altered by mutation  The Genetic Code: DNA – RNA - Protein Genetic code The DNA code that determines how a nucleotide sequence is converted to an amino acid sequence of a protein The Central Dogma of Biology  Alteration of bacterial genes and gene expression can… o lead to the causes of new diseases o prevent disease treatment o be manipulated for human benefit bacterial toxins antibiotic resistance human insulin produced by bacteria  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types  DNA Structure and Replication DNA “alpha double helix” structure o like a “twisted ladder” “rails” – sugar and phosphate backbone “steps” – nitrogenous base; the protein code  DNA – 2 functions 2. Provide “code” for synthesis of proteins Transcription – DNA to mRNA Translation – mRNA to protein 1. Replicate itself Regulation DNA replication o controling gene expression/protein synthesis DNA replication translation: blue-ribosome, red-mRNA, yellow strand – new protein, yellow monomers - tRNA  DNA made of nucleotides o DNA made from building blocks or “repeating subunits” called nucleotides NUCLEOTIDE NUCLEOTIDE nucleotides each nucleotide has 3 parts: “Ladder Rails” Phosphate Sugar (Deoxyribose) “Ladder Steps” Nitrogenous Base (A,T,C or G) DNA – 4 bases; a, t, c, and g  The two DNA strands have OPPOSITE orientations one sugar / phosphate backbone is “reversed” relative to the other DNA strands: 5’ and 3’ ends o Two DNA strands: 5’ END: starts with phosphate one strand starts with phosphate / ends with sugar 3’ END: starts with sugar other strand starts with sugar / ends with phosphate 5’ end: starts with phosphate 3’ end: starts with sugar DNA strand orientation: strands run in “opposite directions” said to run “antiparallel” strand 1: 5’ – 3’ direction strand 2: 3’ – 5’ direction DNA  “Base pairing” rules KEY to replication A-T C-G Remember: A ALWAYS with T C ALWAYS with G o allows each INDIVIDUAL STRAND to serve as TEMPLATE for new strand o each new DNA molecule….. consist of 1 original strand and 1 new strand “Semiconservative Replication” specific base pairing in DNA replication  Steps of DNA Replication STEP 1: o DNA strands relax and separate Enzymes: Topoisomerase, Gyrase and Helicase STEP 2: o DNA nucleotides of EACH single strand are 1. paired with “free-floating” DNA nucleotides Enzyme: DNA Polymerase FINAL RESULT: two new double helices DNA replication steps DNA replication “bubble” - bi-directional replication Important DNA Replication Enzymes o “magical proteins” 1. Topoisomerase and Gyrase 2. Helicase 3. DNA polymerase 4. DNA ligase DNA replication: DNA polymerase - green DNA helicase - blue DNA strands - red Topoisomerase and Gyrase: enzymes “relax” the supercoiled DNA double helix prevent “supercoiling” of unwound strand Topoisomerase and Gyrase Topoisomerase - blue  Helicase: enzyme “unwinds” and “separates” the DNA double helix DNA helicase DNA polymerase DNA polymerase: enzyme that “adds” nucleotides to form the new strand also “proof reads” strand and fixes any mistakes if wrong base added – DNA polymerase backs up, removes misplaced nucleotide and replaces it with the correct one DNA ligase – “seals” sugar/phosphate backbone of nucleotide chain  Energy for DNA Replication is supplied by nucleotides o hydrolysis of two phosphate groups from nucleotide triphosphate provides energy hydrolysis of nucleotide triphosphates provide energy for nucleotide addition in DNA replication  DNA Replication: 5’ to 3’ direction nucleotides added in OPPOSITE directions for each strand strands run “anti-parallel” to each other nucleotides added in 5’ to 3’ direction nucleotides can only be added to 3’ end of the DNA strand 3’ 5’ 5’ 3’ DNA replication – 5’ to 3’ direction  5’ – 3’ strand orientation - creates “problem” for 1 strand Leading Strand: DNA “opens” in 5’ – 3’ direction nucleotides can be added directly; continuous replication DNA replication Lagging Strand: Leading Strand DNA “opens” in 3’ – 5’ direction Lagging Strand 5’-3’ DNA must be added in “fragments” 3’-5’ discontinuous replication small RNA primer added DNA Polymerase: adds nucleotides in “sections” 5’-3’ – removes RNA primers Okazaki Fragment: short DNA sections added in lagging strand synthesis DNA Ligase: seals Okazaki Fragments DNA Replication: enzymes involved in DNA replication DNA Replication Overview: Animation VIDEO: DNA replication  Bacterial DNA replication: o most bacterial DNA replication is bi-directional o each offspring cell receives one copy of the DNA molecule o replication is highly accurate due to the proofreading capability of DNA polymerase bacterial DNA replication bacterial DNA replication  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types  Protein Synthesis o Transcription: DNA to mRNA o Translation: mRNA to protein o Regulation: control of protein synthesis Transcription Translation  Protein Synthesis: 2 steps Step 1: Transcription DNA to mRNA Step 2: Translation mRNA to protein prokaryotic protein synthesis bacteria: can do simultaneous transcription and translation  1. Transcription: DNA to mRNA transferring DNA’s protein code to mRNA transcription 2. Translation: mRNA to protein reading mRNA code and assembling protein 3. Regulation: controlling gene expression and protein production controlling transcription and translation  Transcription Steps - DNA to mRNA STEP 1: DNA strands separate RNA nucleotides added 5’ to 3’ direction STEP 2: RNA nucleotides added U’s will replace T’s on mRNA strand STEP 3: mRNA transcript is released from DNA FINAL RESULT: 1 NEW single strand mRNA RNA Polymerase - important enzyme for transcription Two Functions: 1. “unwinds” and “separates” the DNA double helix Separates the two DNA strands unwinds DNA transcription adds nucleotides 2. “adds” the RNA nucleotides to form new mRNA strand Prokaryotic Transcription  Protein Synthesis: Step 1: Transcription DNA copied to mRNA Step 2: Translation mRNA code converted to protein ribosome tRNA amino acids  Key Molecules Involved in Translation Ribosome - functions as a protein factory converts the genetic information encoded in mRNA into proteins Ribosomal RNA (rRNA) - integral part of ribosome structure. Transfer RNA (tRNA) - transports amino acids to ribosome during protein synthesis Messenger RNA (mRNA) - carries coded information from DNA to ribosomes Amino acid - subunit of protein – assemble at ribosome into proteins Protein - molecule composed of amino acids, resulting from translation Codon - 3 base mRNA sequence that encodes a specific amino acid Anticodon - complimentary aa specific tRNA sequence to mRNA codon sequence  The Triplet Code Codon – 3 base mRNA sequence; codes for specific amino acid; read sequentially Degenerate Code: more than 1 codon for each amino acid 3 Codon types: start stop aa specific codon and the triplet code - DNA to mRNA to protein  The Codon Table The Protein Code: The “Triplicate Code” genetic code universal in all organisms!!! suggests all living organisms have a common evolutionary history!!! mRNA codon to amino acid conversion  tRNA - transfer RNA brings aa’s to ribosome/mRNA complex top binds amino acid tRNA is single stranded: folds and binds with itself by base pairing rules “resembles” double stranded RNA bottom is the anticodon binds mRNA codon  Ribosome’s roll in Translation mRNA: yellow Ribosome: blue tRNA: green Amino acids: red Binds mRNA and serves as “docking station” for tRNA’s carrying aa’s  Treacher Collins (TC) Syndrome: genetic ribosomal disease cause = faulty rRNA gene o TC causes abnormalities in facial bone development Eyes, ears and cheekbones o Can lead to blindness, deafness or suffocation  There are Two Types of Ribosomes: 1. 60s ribosome – prokaryotes, mitochondria and chloroplast – smaller 2. 80s ribosome – eukaryotes (except mitochondria and chloroplast) - larger Ribosome Structure – composed of… ribosomal RNA (rRNA) – catalytic (enzymatic) function protein – structural function ribosome is two subunits: small subunit contains P-site o Prokaryote = 30s subunit o Eukaryote = 40s subunit large subunit contains E and A sites o Prokaryote = 50s subunit o Eukaryote = 60s subunit  Ribosome characteristics: subunits “float separately” in cytoplasm assemble “around” mRNA moves down mRNA strand linking AA’s “disengage” when translation complete large subunit (50s or 60s) small subunit (30s or 40s)  Ribosome: 3 tRNA Binding Sites 1. E-site: Exit site tRNA exit site 2. P-site: Primary site Binds Peptidyl tRNA (tRNA with amino acid chain) 3. A-Site: Addition Site Binds Aminoacytl tRNA (tRNA with incoming amino acid) ACRONYM: “EPA” - Environmental Protection Agency 60s 40s 60s  STEPS of translation: Step 1: Initiation o step that brings all components of translation machinery together small subunit binds start codon 1st tRNA carrying 1st amino acid binds P-site large subunit binds Step 2: Elongation o step where polypeptide grows – 1 amino acid at a time mRNA codon BINDS tRNA anticodon in A-site P-site AA chain transferred to A-site AA – creating empty P-site tRNA Ribosome moves 1 codon empty P-site tRNA to E-site A-site AA chain to P-site P P Now empty A-site… process repeats Step 3: Termination o polypeptide and assembled components are separated from each other. ribosome reaches a stop codon and a protein breaks up ribosome/mRNA complex  STEPS of Translation: Initiation 3 steps of INITIATION: Step 1: Small (40s) ribosome subunit binds mRNA Contains P site Binds at area of START codon Start codon: AUG (always!) Step 2: tRNA with 1st AA binds P site tRNA anticodon binds mRNA START codon AUG start codon codes for aa Methionine (always!) 1st aa of every protein is Methionine!! P P Step 3: Large Ribosomal Subunit Binds Small Subunit Contains E and A-sites Ribosomal complex fully assembled  STEPS of Translation: Initiation Small ribosomal subunit (60s) binds Large ribosomal subunit (60s) binds Contains P-site Contains E and A-sites 1st aa binds Ready for addition of next aa P P P-site P-site  STEPS of Translation: Elongation 4 Steps of ELONGATION: Step 1: 2nd tRNA Binds Codon at A-Site Anticodon binds Codon  Step 2: AA’s Bind Each other Step 3: Translocation Enzyme: Peptidyl Transferase Ribosome “Shifts” 1 Codon Links Amino Acids - Peptide Bond 5’ to 3’ mRNA Direction Attaches P-Site AA to A-Site AA o P site becomes E Site P-Site tRNA / AA Bond Broken o A Site becomes P Site o OPENS new A Site GTPase 5’ 3’  Step 4: 1st tRNA leaves E-Site Elongation Cycle Repeats: o tRNA with next aa binds A-site o Peptide chain binds aa #3 Peptidyl transferase / peptide bond P-site tRNA/AA bond broken o Ribosome shifts again Opens new A-site 3’ 5’ Process continues Elongation Steps of Translation TERMINATION: stopping translation - reaching a STOP CODON on mRNA 1. STOP codon moves into A-site 2. Release Factor Protein NO tRNA for stop codon releases tRNA and protein binds “release factor protein” dissociates ribosomal subunits “release” from mRNA TRANSLATION COMPLETE!!!! Protein Synthesis Overview: Transcription and Translation  many ribosomes can translate single mRNA called “poly-ribosome complex” poly-ribosome complex SEM micrograph: poly-ribosome complex Bacterial Translation: can begin before transcription is complete bacterial translation - can occur during transcription VIDEO: Protein Synthesis - Transcription and Translation VIDEO: Molecular Visions of DNA  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types  operon: a cluster of genes under the control of a single promoter 2 types of operons: o Inducible: normally “OFF” and repressor bound turned “ON” by removal of repressor by an inducer lac (lactose) operon example of inducible operon o Repressible: normally “ON” and repressor not bound turned “OFF” by binding of repressor Trp (tryptophan) operon for amino acid synthesis example of repressible operon We will learn about the INDUCIBLE lac operon! operons – inducible and repressible operon: cluster of genes encoding enzymes for particular metabolic pathway controlled by binding of RNA polymerase to promoter sequence of operon Promoter: DNA sequence – turns gene on/off RNA Polymerase binding site Operator: regulation sequence - DNA sequence binds REPRESSOR PROTEIN lac operon E. coli lactose metabolism Inducible Operon Lactose absent: o Repressor protein: binds lac operator sequence Repressor protein encoded by repressor gene OUTSIDE lac operon o RNA Polymerase: CAN NOT bind promoter RESULT: no enzymes produced for Lactose metabolism  Controlling Gene Expression: Prokaryotes lac operon in E. coli Lactose present: o Lactose BINDS repressor protein Inducible Operon: Genes not transcribed unless inducer is present o Repressor protein can no longer bind lac operator Inducer = Lactose o RNA Polymerase: CAN bind promoter RESULT: enzymes produced for Lactose metabolism VIDEO: lac operon in E. coli VIDEO: lac operon in E. coli VIDEO: lac operon in E. coli  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types  DNA Mutations Mutation: permanent alteration in organism’s DNA Causes: DNA replication error, mutagens (mutation causing agent) Mutation rate very LOW in most organisms 3 possible results: 1.Harmful: Reduces survival rate Mutation removed from population (lower survival) 2.No effect: – MOST mutations 3.Beneficial: Increases survival rate Called “adaptive” mutation (better survival)  two broad types of DNA mutation: 1. Single base substitution 2. Frameshift single base substitution mutation frameshift mutation mutation types: single base substitution vs. frameshift  DNA mutation types Mutation types: o Point mutation (base substitution) Wrong base added in DNA replication Permanent base substitution when strand replicates Causes single faulty codon o Addition mutation Extra base inserted Disrupts ALL codons downstream Frameshift mutation o Deletion mutation Deletion of base in sequence Disrupts ALL codons downstream Frameshift mutation  General Mutation types: 1. Missense: base substitution results in change in an amino acid 2. Nonsense: base substitution results in a nonsense (stop) codon 3. Frameshift: insertion or deletion of one or more nucleotide pairs frameshift mutations shift the translational "reading frame" DNA mutations: missense vs. frameshift  Mutations and Evolution: o Mutations and genetic recombination creates cell diversity o Diversity is the raw material for evolution o Natural selection acts on populations of organisms to ensure the survival of organisms fit for a particular environment  MUTATIONS - some can be BENEFICIAL!!!! sometimes give organisms a better chance at survival and reproduction Evolution of the bird from dinosaurs this is the driving force of EVOLUTION!!! it’s called Natural Selection good mutations increase survival and the passing on of these DNA traits bad mutations decrease survival and decrease chance of passing on those DNA traits  MUTATIONS – some can be BAD! Mutations are a major cause of cancer Causes: Mistakes during DNA replication Environmental causes Chemical or UV light exposure Cell has 3 possible responses to mutation: 1. Cell will fix error – cell OK 2. Cell will recognize it can’t be fixed Will stop dividing – CELLULAR SENESCENSE Commit suicide – APOPTOSIS 3. Cell will continue to divide cancer cell some mutations cause a cell to divide uncontrollably……….. this is a CANCER CELL  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types Prokaryotes: the flow of genetic information from cell to cell Vertical gene transfer o flow of genetic information from one generation to the next o Binary Fission bacterial genetic transfer o horizontal – between cells conjugation transduction transformation o vertically – parent to daughter cells binary fission cell division Horizontal gene transfer o flow of genetic information between cells of same generation o Conjugation, Transduction and Transformation  Horizontal Gene Transfer o three ways gene(s) are transferred INTO bacteria: two separate bacteria methods of bacterial horizontal gene transfer 1. Transformation: uptake of “free-floating” plasmids by bacterial cells 2. Transduction: transfer of DNA into bacteria via a bacteriophage 3. Conjugation: transfer of plasmids between bacterial cells  Transformation: uptake of “free-floating” plasmids by bacterial cells Heat Shock Transformation Method: add CaCl2 salt to solution of bacteria and plasmid place in 42°C water bath for 50 seconds place tubes on ice  steps of transduction Transduction: o transfer of DNA into bacteria by bacteriophage or retrovirus o bacteriophage – virus specific to infect bacteria o viral DNA inserted into cell genome and excised after replication for re-packaging and exit of new virus o inaccurate viral DNA excision can “take” bacterial DNA with it o bacterial DNA is transferred when new virus infects another bacteria and inserts its DNA into the cells genome  Conjugation: o transfer of plasmids between bacterial cells o requires cell-to-cell contact via sex pili conjugation horizontal transfer of plasmid from F+ cell to F- cell F+ cells – carry F-factor gene on plasmid for production of sex pilus enables cell to transfer plasmids horizontally F- cells - cells do not have F-factor plasmid…therefore… can only receive plasmid becomes F+ cell after receives F-factor plasmid conjugation - horizontal transfer of plasmid from F+ cell to F- cell  Ch.8 – Microbial Genetics o Central Dogma; mutation; altering bacterial genes o DNA function; DNA replication o Protein Synthesis – Transcription and Translation o Operons – lacZ operon o Mutations o Prokaryotes – horizontal and vertical gene transfer o Plasmids – three types  plasmids circular “rings” of DNA that exist in nature a plasmid a “naturally existing” small circular DNA molecule naturally taken in and transferred between bacteria once in bacteria, plasmids are replicated, transcribed and translated by bacteria often code for proteins that enhance the pathogenicity or metabolism of a bacterium  3 plasmid types: bacterial a plasmid conjugation o Conjugative (F factor) plasmid : carry genes for sex pili and transfer of plasmid o Resistance (R factor) plasmid: encode antibiotic or toxin resistance o Dissimilation plasmid: genes for improved catabolism (breakdown new molecules)  F factor plasmid with several resistance genes this plasmid also contains an F factor. what does that mean? plasmids: can have resistance genes called R-factor or resistance plasmids genes that give bacteria resistance to toxic molecules antibiotic or other toxin resistance plasmids are called R-factor plasmids VIDEO: bacterial genetic transfer – conjugation, transformation and transduction VIDEO: bacterial genetic transfer – conjugation  Question 1 Which of the following statements about plasmids is FALSE? a. Plasmids typically contain a small number of genes that are useful only in particular environments. b. Plasmids may contain virulence genes that confer disease properties on the bacterium. c. Plasmids are essential for survival of the organism. d. Plasmids can be transferred horizontally from one bacterial cell to another. 85  Question 2 The enzyme that synthesizes a second strand of nucleotides from an existing DNA strand is a. DNA polymerase. b. reverse transcriptase. c. DNA ligase. d. RNA polymerase. 86  Question 3 You discover a mutation that causes the production of a truncated (shorter) nonfunctional protein “A.” What type of mutation is most likely? a. a missense mutation in protein “A” b. a nonsense mutation in protein “A” c. a silent mutation in protein “A” d. a nonsense mutation in RNA polymerase e. a missense mutation in RNA polymerase 87 Question 4 Bacteria can regulate gene expression at a variety of levels. Predict which level of control is the most drastic and difficult to reverse. a. changing the DNA sequence b. control of transcription c. translational control d. posttranslational control 88  Ch. 8a Learning Objectives Define genetics, genome, chromosome, gene, allele, genotype, phenotype. What are the functions of DNA and describe how it serves as genetic information. Describe the structure of a nucleotide Describe the structure of DNA. What is meant by the term “antiparallel strands”? Describe DNA replication. What enzymes are involved, their functions? What’s leading/lagging strand? Why are Okazaki fragments needed. Describe the overall process of protein synthesis. Describe the molecules involved and their functions What are the steps, and what happens in each step? Describe a codon and anticodon. How many types are there for each? How do you read the codon table? Compare protein synthesis in prokaryotes and eukaryotes. Define operon. Describe the parts of an operon and their function. Describe the two operon types and how they work. How does the E. coli lac operon work?  Ch. 8a Learning Objectives Define mutation; Discuss their importance and the three possible results of a mutation. Discuss how genetic mutation provides material for natural selection to act upon. Describe the different mutation types and their potential effect on the final protein. Discuss how mutations can be associated with cancer cells. Describe bacterial gene transfer and discuss its importance in evolution. Differentiate horizontal and vertical gene transfer. Describe 3 methods of horizontal gene transfer and their mechanisms of genetic recombination. Discuss the bacterial requirements for conjugation. Distinguish between a F+ and F- bacterial cell. Discuss how genetic recombination provide material for natural selection to act upon. Describe a plasmid and their functions. Discuss three plasmid types and how they can impact a bacterial colony.