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6: Microbial/Bacterial Growth

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    2727
  • Part 1 DNA replication, Transcription and Translation

    DNA replication

    I. Chromosomal DNA


    A. Function: DNA base sequence encodes information for amino acid sequence of proteins. Genetic code: 1 to 1 relationship between a codon (specific sequence of 3 bases) and 1 amino acid. Central Dogma of genetics/info flow in cells
    -Foundation Figure: Flow of Genetic Info p

    1. DNA will be replicated and passed on to “daughter cells”

    B. DNA Structure: figure 8.3 Double stranded (2 strands of DNA), helical “double helix”, antiparallel


    1. Two strands held together by hydrogen bonds between complementary bases inside helix
    2. Strong outer “sugar-phosphate” backbone; covalent phosphodiester bonds link
    nucleotides
    3. DNA strands: polymers of nucleotides
    4. Nucleotides: 3 components. Sugar=deoxyribose, phosphate, nitrogenous base
    5. Nitrogenous bases of DNA

    a. purines (2 rings)= adenine (A) and guanine (G)
    pyrimidines (1 ring)= thymine (T) and cytosine (C)
    b. Chagraff’s rules: amount of A=T and amount of C=G; this is because of
    complementary base-pairing rules
    A=T form 2 hydrogen bonds
    G=C form 3 hydrogen bonds
    *c. complementary base pairing permits the precise replication of DNA

    6. Deoxyribose: pentose 5 carbons. C1' covalently linked to nitrogenous base.
    C3’= free OH (tail)
    C5’ linked to phosphate group (head)
    7. Prokaryotic chromosomes see figure ; Most bacteria have a single circular chromosome. 1 copy of chromosomes=“haploid cells” (most human cells have 2 copies of linear chromosomes and are called “diploid cells” see “eukaryotic chromosomes ).
    8. Topoisomerases and Bacterial Gyrase
    -Topoisomerases; Enzymes which “supercoil” DNA or relieve supercoiling different types of toposiomerases in E. coli.
    Type I/III” “relax” DNA supercoils
    Type II= Bacterial Gyrase: introduces negative supercoils
    “Through the action of topoisomerases, the DNA molecule can be alternately coiled and relaxed. Because coiling is necessary for packing DNA into the confines of a cell and relaxing is necessary so DNA can be replicated (and transcribed), these two complementary processes ..play an important role in the behavior of DNA in the cell. “
    Brock Biology of Microorganisms 8th edition p 185 )
    -bacterial gyrase is involved in supercoiling/relief of supercoiling of DNA
    -antibiotics quinolones (e.g. nalidixic acid) and fluoroquinolones (such as
    ciprofloxacin) and novobiocin inhibit bacterial gyrase and interfere with
    DNA replication/transcription; see p

    C. DNA synthesis by DNA polymerases fig ___; Table _____

    1. DNA polymerase requires template strand (guide), primer strand with free 3’OH group, activated substrates/precursors= nucleoside triphosphates

    *2. DNA replicated in 5’ to 3’ direction (5’->3’). Incoming nucleotides can only be added to 3’OH tail of a growing DNA strand

    3. Oxygen of 3’OH groups makes a nucleophilic attack on inner most phosphorus atom of incoming nucleoside triphosphate. Pyrophosphate split off and will be hydrolyzed by cellular phosphatases with the release of energy to drive synthesis. Nucleotide is linked to primer strand by phosphodiester bond (ester bond= bond between alcohol and acid)

    4. If no 3’OH present , DNA strand cannot be lengthened=DNA chain termination. Use of dideoxynucleoside triphosphates as base analogues and in DNA sequencing reactions.

    II. Replication of Bacterial Chromosome

    fig ____

    A. Recall bacterial chromosome: singular, circular double stranded DNA in cytoplasm

    B. DNA replication begins at specific site “ori” = origin of replication

    C. DNA replication proceeds bidirectionally from ori, with formation of replication bubble and 2 replication forks. Replication forks= regions where d.s. DNA unwound, form s.s. DNA templates, DNA polymerase makes complementary copy of parent ssDNA template.

    D. DNA replication is semiconservative. 1 parent “old” DNA strand is used as template or guide
    for synthesis of 1 new daughter DNA strand.
    -result: 1 parent chromosome -> 2 daughter chromosomes. Each daughter chromosome is a copy of parent chromosome. Each daughter chromosome consists of 1 old parent DNA strand and 1 new daughter DNA strand. 1 parent strand is “conserved” in each new daughter chromosome

    E. Enzymes/proteins involved in DNA synthesis. KNOW FOR EXAM. Fig 8___ Table ___

    1.* Topoisomerases ex Bacterial Gyrase; involved in DNA supercoiling/relief of
    supercoiling (target of quinolones ex ciprofloxacin “cipro” used to treat/prevent
    inhalation anthrax)

    1. Helicase: unwinds ds DNA, breaks H bonds between bases, forms ss DNA template

    2. Single Strand Binding Proteins SSBP bind, stabilize and protect ssDNA

    3. RNA Primase: an RNA polymerase which does not require a primer strand to start primer synthesis. Synthesizes a short complementary RNA primer strand with free 3’OH group using ss DNA as template. Creates RNA primer, permitting DNA polymerase to start DNA synthesis. ( RNA polymerase do not “proof read” and therefore can make many mistakes).

    4-5. DNA polymerase: requires primer strand, template and activated nucleoside triphosphates (dATP, dTTP,DCTP,dGTP). Must have DNA template. Synthesizes complementary DNA strand using parent strand as template/guide. DNA polymerase have “proofreading abilities”, they “check” each nucleotide they add, remove if incorrect and add correct nucleotide. DNA polymerases have high fidelity, they make very few mistakes. Original mistake rates 10-4; following proofreading, mistake rate= 10-9 ie one incorrect base in every 109 bases added E. coli: DNA polymerase III performs most of DNA synthesis
    DNA polymerase I: will remove RNA primer and replace with DNA sequence

    6. Ligase: links short sequences of DNA (called Okazaki fragments) together on “lagging
    strand” homework see inhibition of nucleic acid synthesis. What are nucleotide analogs? What are their uses?

    Compare and contrast bacterial DNA polymerases and RNA polymerases
    Note: ss=single strand ds=double strand P=phosphate
    Overview:
    DNA polymerases synthesize complementary DNA using a DNA template/guide
    dna
    Ex ssDNA template base sequence: A T A G G C
    Complementary DNA sequence T A T C C G dna
    synthesized by DNA polymerase


    RNA polymerases synthesize complementary RNA sequences using DNA as a
    template/guide
    dna
    Ex ssDNA template base sequence: A T A G G C
    Complementary RNA sequence U A U C C G rna
    synthesized by RNA polymerase


    Synthesis of DNA and RNA require input of energy, both ATP and charged precursors

    Compare and Contrast DNA Polymerase and cellular RNA Polymerase
    ------------------------------------------------------------------------------------------------------------
    DNA Polymerase RNA Polymerase
    Template/guide ss DNA ssDNA
    Synthesize complementary DNA complementary RNA
    Charged precursors deoxyadenosine tri-P= dATP adenosine tri-P= ATP
    deoxythymidine tri-P=dTTP uridine tri-P=UTP
    deoxycytodine tri-P= dCTP cytodine tri-P=CTP
    deoxyguanosine tri-P=dGTP guanosine tri-P=GTP
    primer required? yes no
    proofreading/editing? yes * no
    -------------------------------------------------------------------------------------------------
    *DNA polymerase proofreading/editting
    Polymerases have a ”normal” or “intrinsic” mistake rate of approximately
    10 -4 – 10 -5 nucleotides (this means the polymerases introduce the incorrect nucleotide
    every 10,000 to 100, 000 nucleotides). DNA polymerases have the ability to “proofread
    and edit” their mistakes. If they introduce the wrong nucleotide, they can remove or
    “excise” the wrong nucleotide and try again to make a correct match. This reduces the
    mistake rate of DNA polymerases to approximately 10-9 – 10 -10 (or only one incorrect
    nucleotide every 1,000,000,000 – 10,000,000,000 nucleotides). RNA polymerase cannot
    proofread or edit so RNA polymerase make many mistakes (one reason many
    RNA viruses, for example HIV, mutate so rapidly…..more later)

    Transcription Prokaryotic

    Review flow of information in cell
    DNA--------> RNA --------->Protein

    replication transcription translation


    I. Genetic Code: one to one relationship between specific codon (specific 3 base
    sequence) and an amino acid

    II. Bacterial Transcription: use of DNA as template/guide to synthesize complementary RNA.
    DNA info is rewritten in RNA sequence. Fig ___

    A. First step in gene expression

    B. Products of transcription

    1. messenger RNA=mRNA: will be translated into specific amino acid
    sequence of a protein
    2. transfer RNA=tRNA: actual “translator” molecule, recognizes both a
    specific codon and specific amino acid
    3. ribosomal RNA=rRNA: combined with ribosomal proteins, will form
    the ribosome, the “workbench” at which mRNA is translated into a specific amino acid
    sequence/polypeptide/protein

    4. additional RNA products

    III. Promoters and Bacterial RNA polymerases

    A. Promoters: specific DNA sequences which signal the “start” points for gene
    transcription. Sigma factor/subunit of RNA polymerase binds to promoters to
    initiate transcription


    B. Bacterial RNA polymerases: enzyme complex which recognizes DNA promoters, binds
    to promoter and synthesizes complementary RNA copy using DNA as
    template/guide

    1. E. coli RNA Polymerase: 2 subunits, sigma subunit and core
    a. sigma subunit/factor= “brains” of RNA polymerase. Travels along DNA until it reaches a promoter, binds promoter
    b. core subunit: binds to sigma attached at promoter. “Workhorse” of RNA polymerase, carries out actual RNA synthesis. Requires activated precursors and template strand, DOES NOT REQUIRE PRIMER (compare to DNA Polymerase). Synthesizes RNA in 5’ - to->3’ , similar to DNA polymerase. No proofreading ability therefore will make more mistakes than DNA Polymerase
    c. sigma subunit will drop off after the first few ribonucleotides have been linked together, core continues alone. Note: core would start transcription randomly of DNA without direction of sigma subunit. Polycistronic mRNA (prok. only)

    IV. Termination of transcription (over-simplified)

    A. terminators: DNA sequences which signal transcription stop signals. RNA
    polymerase releases DNA when transcription terminator sequence encountered Homework Describe antimicrobial drugs which bind to and inhibit function of bacterial RNA polymerases (answer: rifampin _used to treat which pathogen?)

    Bacterial Translation fig


    I. Translation: RNA base sequence translated into amino acid sequence of protein. mRNA is template for
    polypeptide synthesis. Second step in gene expression.

    A. Translation of mRNA into a polypeptide chain is possible because of the genetic code:

    1. genetic code: One to one relationship between a codon (specific sequence of 3 bases)
    and a specific amino acid.
    Figure __ Genetic code table

    examples
    mRNAcodon=amino acid
    GAC=aspartate
    CCU=proline
    UUG=leucine
    Genetic code: Redundant (more than one codon for each amino acid) yet specific (each codon
    encodes info for 1 amino acid only). Universal; most cellular organisms use same genetic code;
    some exceptions

    B. Translation requires tRNA, amino acids, ATP/GTP, ribosomes and mRNA

    C. tRNA =transfer RNA. Adaptor/translator molecule. Only molecule which can "recognize"
    correct amino acid AND correct codon

    1. structure: ss RNA, stem and loop
    a. amino acid attachment site at one end
    b. anticodon which "recognizes"(forms H bonds with) codon of mRNA
    2. *45 different tRNA’s for 20 different amino acids; “wobble” permits some tRNA’s to
    bind to more than one codon (“relaxed”/improper base painring between 3 base of codn
    and anticodon)

    D. amino acyl tRNA synthetases* : “load” proper amino acid on proper tRNA= amino acid activation. 20 different transferases for 20 different amino acids/tRNA’s amino acid x+ ATP + tRNAx--> tRNAx:amino acid x + AMP + 2 P* “charged tRNA” or “activated amino acid”

    E. Ribosomes: 70S in prokaryotes. 2 subunits 30S (small subunit) + 50S (large subunit) S=Svedberg Unit, use to express sedimentation rates, ultracentrifugation

    made of rRNA and ribosomal proteins. “Workbench” at which mRNA will be
    translated into a polypeptide. 16s rRNA binds RBS (Ribosomal Binding Site on mRNA). 23s rRNA acts a ribozyme, forms peptide bonds between amino acids
    E, P and A sites.

    F. Mechanics of translation: text. GTP is hydrolyzed during translation

    Translation Initiation (note: tRNA-f met may first bind 30S subunit before 30S subunit binds
    RBS)

    1. 30S subunit recognizes ribosomal binding site RBS/Shine-Dalgarno sequence.
    Complementary to 16s rRNA sequence of ribosome.
    2. Translation begins at start codon AUG closest to ribosomal binding site
    3. An initiator tRNA:methionine ( more precisely a formyl methionine in bacteria) enters
    the “P” or peptidyl binding site of the ribosome. A tRNA fits into the binding site
    when its anticodon base-pairs with the mRNA codon
    4. The larger ribosomal 50S subunit then binds the complex
    5. Additional proteins called initiation factors are required to bring all components
    together

    Translation Elongation: amino acids are added one by one to first amino acid. Additional protein
    elongation factors required

    1. A second appropriately charged tRNA enters the “A” or aminoacyl binding site of the
    ribosome, bearing the next amino acid.
    2. Peptide bond formation. 23s rRNA of large subunit catalyzes formation of peptide
    bond between amino acid at P site and amino acid at A site (rRNA acts as a “ribozyme”, RNA catalyst)
    -amino acid of tRNA at P site is transferred to amino acid bond of tRNA at A site
    3. Now ribosome moves “downstream” by one codon. tRNA carrying dipeptide is now in P site, A site is empty.
    4. New appropriately amino acid charged tRNA enters A site
    5. Ribosome catalyzes peptide bond formation between dipeptide and new incoming
    amino acid. Tripeptide is carried by tRNA at A site
    6. Translocation:
    7. Requires energy (GTP )

    Translation Termination

    1. Ribosome reaches one of 3 nonsense codons/stop codons: UAA, UGA, UAG
    2. Release factor binds A site, causes polypeptide and ribosome to be released from
    mRNA (by activation of ribozyme)

    G. Polycistronic mRNA in prokaryotes permit coordinated gene expression in prokaryotes

    mRNA encodes more than one gene so ribosomes can coordinately produce several
    different proteins. For example 3 genes for proteins involved in lactose transport/metabolism in E. coli are
    transcribed into a single mRNA molecule. Ribosomes translate all 3 into proteins at same time
    H. Simultaneous transcription and translation in prokaryotes only. Ribosomes can bind
    mRNA and begin translation before transcription is finished. Very efficient. Fig ____

    Part 2

    Microbial Genetics: DNA Polymerase, RNA Polymerases. Transcription

    Compare and contrast bacterial DNA polymerases and RNA polymerases

    Note: ss=single strand ds=double strand P=phosphate

    Overview:

    DNA polymerases synthesize complementary DNA using a DNA template/guide

    ___________________DNA

    Ex ssDNA template base sequence: A T A G G C

    Complementary DNA sequence T A T C C G DNA

    synthesized by DNA polymerase

    RNA polymerases synthesize complementary RNA sequences using DNA as a template/guide

    ___________________DNA

    Ex ssDNA template base sequence: A T A G G C

    Complementary RNA sequence U A U C C G RNA

    synthesized by RNA polymerase

    Synthesis of DNA and RNA require input of energy, both ATP and charged precursors (see below)

    ------------------------------------------------------------------------------------------------------------

    DNA Polymerase RNA Polymerase

    Template/guide ss DNA ssDNA

    Synthesize complementary DNA complementary RNA

    Charged precursors deoxyadenosine tri-P= dATP adenosine tri-P= ATP

    deoxythymidine tri-P=dTTP uridine tri-P=UTP

    deoxycytodine tri-P= dCTP cytodine tri-P=CTP

    deoxyguanosine tri-P=dGTP guanosine tri-P=GTP

    primer required? Yes No

    proofreading/editing? Yes* No

    -------------------------------------------------------------------------------------------------

    *DNA polymerase proofreading/editting

    Polymerases have a ”normal” or “intrinsic” mistake rate of approximately

    10 -4 – 10 -5 nucleotides (this means the polymerases introduce the incorrect nucleotide every 10,000 to 100, 000 nucleotides). DNA polymerases have the ability to “proofread and edit” their mistakes. If they introduce the wrong nucleotide, they can remove or “excise” the wrong nucleotide and try again to make a correct match. This reduces the mistake rate of DNA polymerases to approximately 10-9 – 10 -10 (or only one incorrect nucleotide every 1,000,000,000 – 10,000,000,000 nucleotides). RNA polymerase cannot proofread or edit their work so RNA polymerase make many mistakes (one reason many RNA viruses, for example HIV, mutate so rapidly…..more later)

    Transcription Prokaryotic repeated section

    File:C:/Users/Karen/AppData/Local/Temp/msohtmlclip1/01/clip_image001.pngReview flow of information in cell

    DNA--------> RNA ---------> Protein

    replication transcription translation

    I. Genetic Code: one to one relationship between specific codon (specific 3 base sequence) and an amino acid

    II. Transcription: use of DNA as template/guide to synthesize complementary RNA. DNA info is rewritten in RNA sequence.

    A. First step in gene expression

    B. Products of transcription

    1. messenger RNA=mRNA: will be translated into specific amino acid sequence of a protein

    2. transfer RNA=tRNA: actual “translator” molecule, recognizes both a specific codon and specific amino acid

    3. ribosomal RNA=rRNA: combined with ribosomal proteins, will form the ribosome, the “workbench” at which mRNA is translated into a specific amino acid sequence/polypeptide/protein

    III. Promoters and RNA polymerases

    1. Promoters: specific DNA sequences which signal the “start” points for gene transcription. Sigma factor/subunit of RNA polymerase binds to promoters to initiate transcription

    B. RNA polymerases: enzyme complex which recognizes DNA promoters, binds to promoter and synthesizes complementary RNA copy using DNA as template/guide

    1. E. coli RNA Polymerase: 2 subunits, sigma subunit and core

    a. sigma subunit/factor= “brains” of RNA polymerase. Travels along DNA until it reaches a promoter, binds promoter

    b. core subunit: binds to sigma attached at promoter. “Workhorse” of RNA polymerase, carries out actual RNA synthesis. Requires activated precursors and template strand, DOES NOT REQUIRE PRIMER (compare to DNA Polymerase). Synthesizes RNA in 5’ -to->3’ , similar to DNA polymerase. No proofreading ability therefore will make more mistakes than DNA Polymerase

    c. sigma subunit will drop off after the first few ribonucleotides have been linked together, core continues alone. Note: core would start transcription randomly of DNA without direction of sigma subunit. Polycistronic mRNA (prok. only)

    IV. Termination of transcription

    1. terminators: DNA sequences which signal transcription stop signals. RNA polymerase releases DNA when transcription terminator sequence encountered

    Part 3

    Microbial Genetics

    Prokaryotic Regulation of Genetic Expression

    Regulation of transcription: Operons and operators

    Vocabulary

    structural genes: genes which encode information for a protein

    “gene expression”= transcription (+ translation when describing structural genes)

    -for our class discussion, we will presume that when a structural gene is transcribed, the mRNA will be translated into protein thus “gene expression” results in protein production

    -ase= common ending for enzymes

    lactose: also called “milk sugar”, lactose is a disaccharide of glucose and galactose joined by a glycosidic

    bond. lactose= galactose-glucose

    promoter: special DNA sequence to which RNA polymerase binds to start transcription

    operon: DNA sequence encoding promoter and operator sites and structural genes they control

    operator: special DNA sequence to which repressor proteins may bind to block transcription by RNA

    polymerase; the “on-off” switch for transcription

    allosteric proteins: proteins which can have 2 different forms

    lac repressor protein: an allosteric protein which has 2 forms:

    1. an active form in which the repressor bind to the lac operator and blocks transcription of the lac operon structural genes when lactose is absent and …
    2. …an inactive form which cannot bind the lac operator. The inactive form is dominant when lactose is present and the inducer allolactose binds to the lac repressor protein.

    Intro survival notes:

    Gene expression and protein synthesis are costly to cells (requires lots of “building blocks” and

    energy.

    (From Bauman) ….most bacterial genes are expressed constantly= constitutive expression. These genes include genes for tRNAs, rRNAs and structural genes for proteins constantly needed for example, ribosomal proteins and enzymes used in glycolysis.

    However some gene products may be required only under certain conditions. For example, the gene encoding the enzyme beta-galactosidase need only be expressed if lactose is present in the environment. Beta-galactosidase is the bacterial equivalent of human “lactase”. It catalyzes hydrolysis of the glycosidic bond between the residues of glucose and galactose in milk sugar, lactose:

    Lactose-> beta-galactosidase -> glucose + galactose

    Bacteria which can turn on and turn off transcription of genes like beta-galactosidase will have a survival advantage over other bacteria which cannot regulate gene expression (Why?)

    Regulation of Bacterial Gene Expression: transcription control

    1. 3 types of gene expression

    -constitutive : continual transcription (and continual synthesis of encoded proteins)

    -inducible : made only when substrate or signal molecule is present ex enzymes for lactose transport/metabolism in E. coli

    -repressible : produced only when signal molecule is scarce

    2. . Operon: group of genes whose transcription is coordinately turned on or off; under control of operator and promoter

    3. Operator: specific DNA sequence which lies between promoter and 1st codon of gene. Repressor proteins bind operator and block ability of RNA polymerase to bind promoter/ transcribe “downstream” structural genes. Repressor proteins are allosteric proteins. The have one binding site for a DNA operator sequence and a second binding site for an “inducer” molecule for e.g. allolactose in the lac operon ( or a “corepressor” molecule e.g. trp operon )

    C. Inducible operons. Gene transcription turned only when substrate/signal is present. Usually genes for catabolic enzymes example: E. coli lac operon. Jacob and Monod 1961

    1. Lac operon consists of 3 structural genes (lacZ, Y and A) , promoter and operator.

    Lac operon is inducible.

    Structural gene gene product (enzyme/transport protein)

    lacZ beta-galactosidase

    lacY lactose transport protein/ galactoside permease

    lacA galactoside transacetylase

    -----------------------------------------------------------------------------------------------------

    2. Regulatory/repressor gene, lacI

    LacI is the lac repressor protein.

    The lac repressor protein is a DNA binding protein, which when active can bind to the lac operator, blocking transcription/expression of lacZ, Y and A in absence of lactose (survival note: it would be wasteful for the bacterium to make these proteins if lactose is not present) . Lac repressor gene has its own promoter and is constitutively expressed. The lac repressor gene is NOT part of the lac operon

    3. lac operon when NO LACTOSE AVAILABLE (fill-in cartoon below, double dotted lines represent a single strand of DNA)

    DNA ___________________________________________________________

    ------------------------------------------------------------------------------------------------

    lac I Promoterlac Operatorlac lacZ lacY lacA

    How should your cartoon look?

    lac repressor protein binds lac operator in absence of lactose, blocks transcription of lac genes by RNA polymerase. Note: all repression is “leaky”, that is repressor binds and releases operator in a concentration dependent manner. When lactose is absent, most repressor proteins are in the active form, blocking most (but not all) transcription of the lac structural genes)

    4. ADD LACTOSE: (How does your cartoon differ from above?)lactose-> inducer allolactose binds to allosteric site on lac repressor, causes it to change shape so it can no longer bind operator.

    DNA ___________________________________________________________

    ------------------------------------------------------------------------------------------------

    lac I Promoterlac Operatorlac lacZ lacY lacA

    DNA ___________________________________________________________

    ------------------------------------------------------------------------------------------------

    lac I Promoterlac Operatorlac lacZ lacY lacA

    5. Now RNA polymerase can start transcribing the lac genes and cell can makes transport protein and beta-galactosidase, cell starts transporting and breaking down lactose at high rate

    6. When lactose used up, allolactose levels drop/release lac repressor, repressor regains shape, binds operator, turns off lac gene transcription

    DNA ___________________________________________________________

    ------------------------------------------------------------------------------------------------

    lac I Promoterlac Operatorlac lacZ lacY lacA

    What would happen if…..

    What if….

    E.coli had a mutation so that the lac repressor could not bind DNA?

    “ “ had a mutation so that the mutant lac repressor protein could NOT bind allolactose, the inducer?

    “” had a mutation so that the operator could not bind normal lac repressor protein?

    Deleted section from previous semesters:

    I. Regulation of metabolism: 2 ways

    A. Change activity of enzymes: allosteric sites/allosteric enzymes

    1.binding sites other than active site=allosteric sites, bind “effectors”

    2. binding of effector changes 3-D shape of enzyme

    3. 2 kinds of effectors:

    a. allosteric activators: bind allosteric site, change enzyme so that it is MORE ACTIVE/turned on/activate enzyme

    b. allosteric inhibitors: bind allosteric site, change enzyme shape so it is less active/turned off /inhibit enzyme

    ex: end product inhibition

    4. Provides rapid response to changes in substrates, need for endproducts

    B. Change expression of genes that is regulate transcription (or even translation)

    Mutations

    Evolution and Natural Selection

    Unity and Diversity of Life

    All cellular organisms share certain traits as a result of sharing (or “descending from”) a common ancestor (the first primitive prokaryotic cells). Yet the incredible diversity of living organisms is astounding. How did such diversity arise?

    The diversity of life is explained by evolution, a change in the genetic makeup of a population of organisms.

    The “raw material” of evolution is genetic variability, the genetic differences between individuals of a population of organisms. How does genetic variability arise?

    Genetic variability arises through random mutations, changes in the nucleic acid sequence of organisms, horizontal gene transfer in microbes and sexual recombination.

    Random mutations: These mutations usually occur when the enzymes which copy genetic information before a cell divides make a mistake. As an example, the enzyme DNA polymerase should copy the DNA sequence below precisely.

    DNA base abbreviations:

    A=adenine T=Thymine G=Guanine C=cytosine

    Original genetic info, DNA base sequence of double stranded DNA

    A-T- C- G- G

    T- A- G- C- C

    Original DNA

    DNA polymerase should make a precise copy of the DNA before the cell divides such that each “daughter” cell receives a precise copy of genetic information from the parent cell:

    A-T- C- G- G

    T- A- G- C- C copy 1

    A-T- C- G- G /

    T- A- G- C- C --- DNA polymerase copies

    Original DNA \ A-T- C- G- G

    T- A- G- C- C

    copy 2

    When the original cell divides, both “daughter” cells will receive one copy of the DNA, exact copies of the DNA from the “parent” cell

    One source of genetic variability occurs when DNA polymerase makes a mistake copying the original DNA as shown below:

    A-T- C- G- G

    T- A- G- C- G* ,<-mistake=mutation copy 1

    A-T- C- G- G /

    T- A- G- C- C --- DNA polymerase copies

    Original DNA \ A-T- C- G- G

    T- A- G- C- C

    copy 2

    In this case, DNA polymerase made a mistake in copy #1 (instead of a “C”, it used a “G”). This is a mutation, a change in the DNA sequence. As a result, the mutated gene might encode information for a different variety of protein. This different protein could change some feature of the “daughter cell” which receives the mutant DNA copy. This mutant organism might be a better competitor compared to “normal, non-mutant” member of its population (or the mutant might be less able to compete with its “normal” colleagues).

    If resources are limited, “variants” or mutants which are better competitors will survive in higher numbers than their non-mutant neighbors. The mutants will have more offspring than the non-mutant members of the population which are weaker competitors. The mutation which permitted the variant to compete better will be passed on to its offspring. Over time, the mutant form of the genetic information will be carried by a greater proportion of the surviving population, another way of saying the frequency of the mutant gene increases in the population. Thus the gene pool or genetic makeup the population has changed over time and the population of organisms has “evolved” (and the population becomes better adapted to the environment over time) .

    This mechanism above describing “how” evolution occurs is called “natural selection”, a concept developed by Charles Darwin and Alfred Wallace in the 1800’s. (Darwin and Wallace were not the first people to describe evolution, but they were the first describe the process of natural selection).

    Darwin and Wallace’s explanation of evolution by natural selection is based on 5 assumption (source: Keeton and Gould’s Biology 4th edition, Norton Publishers)

    1. Many more individuals are born in each generation than will survive and reproduce (limiting environmental resources, competition for resources)

    2. There is variation among individuals of a population.

    3. Individuals with certain characteristics have a better chance of surviving and reproducing than individuals with other characteristics.

    4. Some of the differences resulting in differential survival and reproduction are heritable

    5. Vast spans of time have been available for change

    Natural selection vs Artificial selection

    When “nature” chooses which variants are best at competing for natural resources, and thus will survive and reproduce at higher rates than other variants , “natural selection” occurs. However if humans selectively breed organisms for specific traits, or if humans purposely change the environment (for example through overuse of antibiotics), “artificial selection” occurs. The overuse/misuse of antibiotics worldwide has artificially selected for a growing number of antibiotic resistant bacteria.

    Evolution of antibiotic resistant bacteria

    As bacteria reproduce so quickly, it does not take “vast spans of time” for their populations to evolve. Antibiotic resistance can evolve within a few years (even within a few weeks) within some populations of bacteria.

    Evolution of variant strains of RNA viruses: HIV and influenza

    Cellular organisms all have DNA as their genetic information. The enzyme which copies DNA, the DNA polymerases, have “high fidelity”, that is these enzymes make relatively few mistakes because they have the ability to “edit or proofread” their work and correct many of their mistakes. Mistake rates fro DNA polymerase are approximately one incorrect nucleotide per 108-109 nucleotides.

    In contrast, some types of viruses (acellular pathogens) use RNA as their genetic information. The enzymes which copy RNA, the RNA polymerases, lack the ability to correct mistakes, therefore RNA polymerases have a very high mistake rate (one incorrect nucleotide every104-105 nucleotides) . Consequently many RNA viruses such as influenza virus and HIV, have very high mutation rates thus “populations” of HIV and influenza viruses evolve quickly. The high mutation rate of these viruses results in rapid drug resistance and huge challenges in vaccine production.

    Natural Selection in a “nutshell”

    1. Each species produces more offspring than can survive

    2. The offspring compete with one another for limited resources

    3. Organisms in every population vary

    4. Organisms with most favorable traits/variations are most likely to survive and produce more offspring

    Result:…..consequently variant genes “spread” through the population over time, genetic makeup of population changes and population changes/evolves over time=”Evolution”

    Horizontal Gene Transfer in Bacteria

    Transfer of genetic material between prokaryotes: transformation, transduction, conjugation

    I. Genetic Recombination and Homologous Recombination an exchange of DNA sequences by crossing over- permits integration of “foreign” homologous DNA into bacterial host chromosome. Replaces bacterial alleles w/ “foreign” alleles.

    1. “Foreign” single stranded homologous chromosomal DNA aligns with homologous sequences of host bacterial chromosomal DNA

    2. DNA fragment of host DNA excised, replaced by foreign DNA sequence-alters cells genotype (“native” allele is replaced w/ a foreign allele)

    3. Requires many enzymes including recombination enzymes/proteins, nucleases, ligases

    II. Transformation: uptake of “naked” DNA from environment by competent bacterial cells.

    1. plasmid DNA or chromosomal DNA

    2. Some bacterial strains naturally competent. Special surface proteins recognize and take-up closely related naked DNA from environment (recall Griffith’s experiments w/ Streptococcus pneumoniae). Other bacterial strains can be made competent by treatment with chemicals (ex cold + calcium chloride treatment used in lab to create competent E. coli cells; permitted uptake of double stranded plasmid DNA)

    3. Competent Streptococcus pneumoniae permit passage of single stranded DNA through cell membrane (2nd strand is usually degraded).

    4. When competent cell is transformed with DNA, the cells are referred to as “transformants. Chromosomal DNA may undergo genetic recombination with bacterial DNA if homologous sequences are present.

    III. Transduction. DNA transfer process in which bacteriophages carry DNA from one bacterium to another bacterium. 2 types: generalized and specialized.

    1. Generalized transduction:(bacterial genes transferred at random) Recall lytic reproductive cycle. Towards end of cycle, bacterial chromosomal DNA is accidentally packaged into phage heads/capsids instead of phage DNA. This creates a defective phage as it lacks its own genetic material, yet it can still be released, attach to new bacterium and inject the DNA into a new host bacterium. This DNA may replace the homologous region of the bacterial chromosome.. The bacterial cell now carries recombinant DNA.

    2. Specialized transduction (only specific bacterial genes are transferred). Requires temperate bacteriophage. Phage DNA integrates into host bacterial chromosome usually at specific site. Later prophage may be induced to enter lytic cycle. When prophage excises from chromosome, sometimes take small stretch of adjacent bacterial DNA. Bacterial genes are packaged w/ phage DNA and injected into new bacterial host.

    IV. Conjugation. Direct transfer of genetic material between 2 bacteria which are temporarily joined. Model uses E. coli (gram positive bacteria use slightly different process).

    1. One-way transfer of DNA: donor cell (male) transfers genetic material to recepient cell (female)

    2. Male uses protein appendages, hollow sex pili, to attach to female

    3. Temporary cytoplasmic bridge forms between 2 cells, providing path for transfer of DNA

    4. “Maleness’, ability to form sex pili and transfer DNA during conjugation, results from presence of special DNA sequence called F factor=Fertility Factor . F factor may exist integrated into chromosome or as a plasmid, therefore it is an episome (episome= genetic element which can replicate either as a plasmid or as part of bacterial chromosome; temperate viruses such as lambda also qualify as episomes).

    5. Recall plasmids are extrachromosomal, self-replicating DNA elements which usually carry “extra’ genes. These genes can confer survival advantages for bacteria living under stressful conditions. For example, “R plasmids” are plasmids carrying genes for antibiotic resistance and permit host cells to survive in presence of antibiotic pressure. F factor facilitates genetic recombination which could be advantageous in a changing environment which no longer favors existing strains of bacteria (Campbell, Biology 5th ed)

    6. F plasmid: The F factor in its plasmid form is called the F plasmid. Consists of approx. 25 genes, most involved in sex pilus production.

    a. F+ cells (males) contain F plasmid; F plasmid is usually replicated and passed to daughter cells. F+ condition is “contagious” as it can be passed to female cells, converting them to F+ males following conjugation

    b. F- cells (females) lack F plasmid

    c. F plasmid is replicated in male and a copy is passed to female, converting her to F+ male. Only F plasmid copy is transferred during F+ x F- conjugation

    7. Hfr and bacterial gene transfer during conjugation

    a. if F factor is integrated into bacterial chromosome, bacterium is referred to as Hfr cell (High frequency of recombination)

    b. Hfr acts as a male, forms sex pilus, copies F factor, starts to transfer copy of F to F- partner, yet now F factor also takes a copy of some of bacterial chromosomal DNA with it

    -temporarily, female is diploid until genetic recombination occurs and segments of DNA are exchanged (excised DNA is degraded)

    -Female becomes recombinant cell; usually mating is interrupted before entire chromosome and F factor are transferred therefore she usually remains female

    V. Resistance Plasmids and Transposons

    1. R =resistance plasmids, carry genes for antibiotic resistance and/or resistance to heavy metals e.g. mercury resistance. Example enzymes to destroy antibiotics. beta-lactamases destroy beta-lactam ring of penicillin, ampiciilin and related antibiotics, mercury reducatase genes.

    2. R plasmids may carry multiple antibiotic resistance genes and can be copied and transferred between bacteria via conjugation and transformation

    3. Some R plasmids carry 10 antibiotic resistance genes; evolution thought linked to transposons

    4. Transposons are transposable genetic elements, pieces of DNA which can move from one location to another (Barbara McClintock’s “jumping genes”1940’s-50’s; Nobel prize 1983 age 81). Some say transposons (“Tn’s”) never exist independently some gram-positive Tn’s may violate this rule.

    5. Transposons may have moved multiple antibiotic resistance genes to R plasmids

    6. Insertion sequences (IS)-simplest transposons. Carry 1 gene only for enzyme transposase bracketed by inverted repeats, upside down, backwards versions of each other . Transposase binds to inverted repeats and recognizes target sites. Transposase cuts target sequences and inserts IS. “Cut and Paste Transposition”

    7. Composite transposons: Additional genes ex for antibiotic resistance sandwich between 2 IS. Probably involved in evolution of R plasmids

    Mutations Worksheet

    Read Ch 8 Tortora p ; see end of handout for good website and some summary notes

    1. What is a mutation?

    2. Describe the following and provide a specific example of each.:

    a. point mutation/base substitution

    b. silent/neutral mutation

    c. missense mutation

    d. nonsense mutation

    e. frameshift mutations

    3. Which of the mutations listed above is potentially most harmful to cells? Why?

    4. a. What are “spontaneous mutations”?

    b. What is the approximate spontaneous mutation rate in a cell?

    c. In contrast, what is the approximate spontaneous mutation rate in an RNA virus?

    5. Upon replicating DNA, cells have 2 initial ways to “fix mistakes”, editing/proofread and mismatch repair. Describe each below

    a. editing/proofreading

    b. mismatch repair.

    - in mismatch repair, how do repair enzymes know which is the correct template strand and which strand needs to be repaired? (answer: the “older” template strand would be methylated, see description of methylases on p 231. The newly synthesized strand would not be methylated (yet). The repair enzymes (called “exinucleases” or more generally “endonucleases”) consequently cut out the portion of the unmethyated strand which does not correctly base pair with the methylated template strand. DNA Polymerase I then replaces with correct DNA, ligase covalently links repaired section with remainder of DNA strand)

    5. What are mutagens?

    Radiation as a mutagen p230

    6. What is non-ionizing radiation? Ionizing radiation?

    7. DNA absorbs maximally electromagnetic radiation of wavelength λ = _____nm. (answer λ =260 nm)

    8.How does ultraviolet (UV) radiation cause DNA damage? Draw and label a cartoon to help illustrate your answer. Draw and label thymine dimers

    9. Bacterial cells have 3 ways to repair DNA damage caused by UV irradiation, specifically repairing thymine dimers. Describe each below ( see notes towards end of handout).

    a. light repair/photoreactivatioin by photolyase: covalent bond of thymine dimer is hydrolyzed in presence of light

    b. dark repair= nucleotide excision repair (used to repair wide range of damage to DNA)

    -thymine dimer and surrounding nucleotides are cut out/excised, gap is filled by DNApolymerase I and linked by ligase.

    c. SOS response: highly error prone repair (“last ditch effort “ to prevent cell death in bacteria)

    10 What is the “Ames Test”?

    11. Explain how mutations may lead to antibiotic resistance.

    12. Human mutations may increase resistance to some infectious diseases. Humans which carry the mutant gene, the “sickle cell allele” , may be more resistant to death caused by _____________- and humans which carry the cystic fibrosis allele may be more resistant to death caused by _________________ (fill-in blanks)

    see: http://www.web-books.com/MoBio/Free/Ch7G.htm

    DNA Repair Mechanisms (from site above)

    Repair system

    Enzymes/proteins

    notes

    “editing/proofreading”

    DNA polymerases

    Mismatch repair (repairs mismatched bases not corrected by proofreading)

    -Dam methylase, Mut proteins, exonuclease, DNA polymerase I, DNA ligase

    Immediately after replication, the template DNA strand has been methylated (by Dam methylase in E.coli), but newly synthesized strand is not methylated yet. Thus the template strand and new strand can be distinguished

    Repair to damaged/altered DNA

    -Repair to damaged/altered bases = “Base excision”

    DNA bases may be modified/altered by deamination or alkylation. In E. coli, DNA glycosylases can recognize altered bases and cut out base only, creating an “abasic site” called the AP site . AP endonucleases remove the nucleoside at the AP site and surrounding nucleotides, gap is filled in by DNA Polymerase I and finished by DNA ligase

    Repair to damaged DNA

    UV damage, thymine dimers

    “light repair”/”photoreactivation”

    Light repair, photolyase

    DNA damage causing distortions e.g. UV damage, thymine dimers

    “dark repair”= nucleotide excision repair

    -Uvr proteins/endonucleases (cut out thymine dimer + 12 nucleotides), DNA Polymerase I, DNA ligase

    SOS repair: response made in “life or death” situation, activated when so much DNA damage that DNA synthesis stops

    Highly error prone

    SOS proteins/enzymes

    Rec A: binds to damaged DNA to initiate recombination repair

    DNA polymerases IV/V (also called “DNA mutases”), which lack proofreading and will synthesize DNA to fill gaps when template is missing

    Generates many many mutations

    update 1.3.2016 K. Carberry-Goh DVM, PhD Sac City College