Bio120 LecPPTS Week 10 11

8/7/2019 Bio120 LecPPTS Week 10 11

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Bacterial Genetics

Review- Eukaryotic

1. DNA structure2. RNA structure

3. DNA replication

4. RNA transcription5. Protein translation


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Genome-all the genes present in a cell or virus;

procaryotes normally have one set of genes (haploid)eucaryotic microbes usually have two sets (diploid)

Genotype-the specific set of genes an organism possesses

Phenotype-the collection of characteristics of an organismthat an investigator can observe

Vertical transmission-transmission of genetic information

from parent to offspring

Horizontal transmission-genetic information transferred from

one cell to another


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1928- Griffith


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Avery, MacLeod, and McCarty

DNA is the genetic material


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tRNA

rRNA

codon

ribosomes

Vertical transmission


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Figure 8-4

Genome Transcription and Translation

Archaea ArchaeaEukarya Eukarya

Bacteria Bacteria

1

4

6 9 10

11 12 1315

1718

195

168

3 14

4

2 7

7 8

11

12 15 143

619

1716

915

2 10

13 18

4 TATA box and BRE sequencein promoter

1 Chromosome circular

versus linear2 Single chromosomeversus multiple

chromosomes3 Introns rare4Archaeal type introns

5 Inteins6 Histones7 DNA gyrase8 Reverse gyrase9 Multiple chromosomal

origins10 Eukaryotic origin

recognition complex

11 Eukaryotic type helicase

19 Telomeres and telomerase

12 B family DNA polymeraseis major replicative

enzyme13 Eukaryotic type sliding

clamp14 Restriction enzymes

15 RNAi16 Genome of double

stranded DNA17 Multiple retroelements

in genome

18 Centromeres19 18S, 28S and 5.8S rRNA

1 RNA used as a geneticmessenger

2 Polycistronic mRNA3 Cap and tail on mRNA

6 Multiple RNA polymerases

5 Repressors binding directly to

DNA in promoter

7 RNA polymerase II with 8 ormore subunits

8 Multiple transcription factorsneeded

9 Ribosomes synthesize proteins1070S versus 80S ribosomes

11 Ribosomal RNA sequencehomologies

12 Ribosomal proteinsequence homologies

13 Shine Dalgarno sequences14 Multiple translation factors15 Elongation factor sensitive to

diphtheria toxin16 N-formyl methionine versus

methionine17 tmRNA rescues stalled

ribosomes18 16S and 23S rRNA


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Table 7-1


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Minimal genome (480 proteins)

Bacterial chromosomes- 517kB-9400kB

Archaeal chromosomes-935kB-6500kB


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Fig.7-10

Parental strand

New strand

Semiconservative

replication

Initiation

Elongation

Termination

OriC

- origin of replication


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Transcription

RNA polymerase and promoters

mRNA-encode proteins

rRNA- ribosomes

tRNA-bind amino acids

sRNA-regulate gene expression, have catalytic activity,

or function as a combination of tRNA and mRNA

Transcription and translation in prokaryotes are coupled.


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OperonsDNA control

sequence

Structural gene-functional RNA molecule


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Eukaryotes

90% of DNA may be noncoding

Prokaryotes

Less that 15% DNA may be noncoding

Splicing

Remove introns

TailingAdenylic acid is added to the 3 end to

produce a polyA sequence about 200

nucleotides long (polyA tail)

Capping

7-methylguanosine is added to the 5

end (5' cap)


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Translation

Start codons AUG GUG UUG CUG

Stop codons UAA UAG UGA

Prokaryotes

30S

50S

Eukaryotes

40S

80S


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Extrachromosomal elements

1. Replicate autonomously within cells

2. Transferred between cells


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Table 11-1


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Plasmids as vectors for cloning


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Genome sequencing

-bacterial and archaeal genomes

-analysis of these genome sequences

ORFs function based on biochemical or genetic studies

large number of hypothetical ORFs with no known function

.


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3D - protein sequence has a 3D model built by homology, or a known 3D structure.

clear_function - protein sequence has functional annotation derived from probable homologues.

tentative_function - protein sequence has functional annotation derived from tentative

homologues

homologue - protein sequence has probable homologues of unknown function

no_homologue - protein sequence has no probable homologues.

E. coli


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Week 11


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In the experiment which proved that DNA was the genetic

material, the method of


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How do bacteria exchange genetic material?

1. Transformation-donor DNA in the environment is taken up by the bacteria

2. Transduction-donor DNA uptake is mediated by a virus

3. Conjugation-transfer involves cell-to-cell contact and a conjugative plasmid


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1. Transformationcompetence-a cell that is able to take up a molecule of DNA and be transformed

artificial competence-induction of competence by treating bacteria with certaincompounds under specific temperature or other factors

electroporation-treating cells with pulsed electric fields to open small pores in themembranes

transfection-transformation with DNA from virus


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Why do species undergo natural transformation?

a. Scrounge DNA from dead cells

b. Use DNA released from dead cells to repair own damaged genomes

c. Adjust to new environment by acquiring new genes


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Gram-positiveStreptococcus pneumoniae

translocasomea. Binding proteinb. Transmembrane

porea. Nuclease

Gram-negative- Must pass through outer membrane


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Conjugative plasmideg. F (fertility) plasmid (F factor)-transferable-circular DNA (99 159 bp)-contain genes needed for pilus formation and DNA export-can integrate into chromosome of host: Episomes

Curing-elimination of plasmids from host cellsConjugation- cell-to cell transfer of plasmids


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Sex pilus-acts as recognition molecule to locate sensitive cell (plasmid minus).-when + and - cell are attached by pilus, cells pulled together


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Conjugation (mating)


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Hfr strains


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Bacterial pheromones-chemicals that promote conjugation

Eg. Aggregation substance produced by pAD1 ( E. faecalis)catalyzes cell-cell contact

-potential recipients secrete small peptides that enter donor cells andstimulate transcription of pAD1 transfer genes

Microbial transfer of genes into eukaryotesEg. Agrobacterium tumefaciens- tumor inducing plasmid (Ti) that is

transferred to plant cells


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2. Transduction

DNA is transferred from cell to cell through a virus (bacteriophage)-amount of genes limited by what can fit in the phage head

Generalized transduction-take any gene from a donor cell and transfer it to a recipient cell(phage cannot distinguish between own DNA from host DNA)

Specialized transduction (temperate viruses)-can only transfer a few closely

linked genes between cells


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Generalized transduction (P1 phage)-virus must have a DNA packaging mechanism that allows accidental recognitionof host DNA


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Specialized transduction-linear phage-recircularizes at cohesive ends(cos sites)

-attP can combine with attB(located between gal and biogenes)-PROPHAGE

-reactivation of prophage

improper excision

-defective phageviral geneshost genes adjacent to the attB

phage


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Site-specific recombination

-requires very little sequence homology between the recombining DNAmolecules but does require a short (10-2bp) sequence recognized by the

recombination enzyme

(integrase)

f


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pSH2

fdh

M. smegmatis

cellHyg resistant

7H10 + Hyg

7H10 Hyg + Kan

pHINT

Hygrfdh

pSH1

Hyg & Kan resistant

Site specific mutagenesis


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Mutation: inherited change in the base sequence of the DNA comprising thegenome of the organism (mutant)

a. harmfulb. beneficialc. neutral

-Cell must fail to repair the change before replication

-genotype: the genetic makeup of an organismeg. hisC gene HisC proteinmutations is hisC: hisC1, hisC2

phenotype: the observable characteristics of an organism(could be different from parent strain)His+ His-

wildtype strain: strain isolated from nature


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Isolation of Mutants

-selectable mutations- confer an advantage to the organism; progeny

outgrows parent strain eg. Drug resistance-nonselectable mutations- may not result in a change in phenotype

Screening

Replica plating

Auxotroph


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Molecular basis of mutationA-spontaneousRates of mutations (DNA genomes)

errors in DNA replication- 10-7- 10-11/round of replication

transposition- 10-7

nonsense mutant- 10-6- 10-8

hot spots

Rates of mutations in RNA genomes-1000 fold higher - no RNA repair mechanisms


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P


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Point mutations-involving one or few base pairs (base pair substitution)Consequences?


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Transition-Purine to a different purine [ A to G ]-Pyrimidine to a different pyrimidine [ C to T]

Transversion-purines to pyrimidines or vice versa[C/ T A / G]

Missense mutationsLoss of function mutation

-decrease or eliminate activity of protein

Gain of function mutation-increase activity-expand substrate specificity


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Mutant 164 is mutated in mshC

164 CTCGACGCGTTG CCG TGGCGCGCCGAGCGTCC

WT CTCGACGCGTTG CTG TGGCGCGCCGAGCGTCC

nucleotide 554

ctg-leucine ccg-proline

5% ACTIVITY

n-METHYL n-NITROSOGUANIDINE


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Insertions and deletions-makes the sequence either longer or shorter-if the number of bases inserted or deleted is not a multiple of three,ribosome will read the wrong triplets

-premature stop codon-different protein sequence

Inversion-occurs when a fragment of DNA is

flipped in orientation relative to theDNA on either side


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Revertant-strain in which wild-type phenotype lost in the mutant is restored1. Same site revertant

2. Second site revertant-suppressor mutationa. a mutation somewhere else in the geneb. a mutation in another gene can restore the

enzyme functionc. a mutation in another gene can result in

another enzyme that results in a new metabolic pathway


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B.Inducedmutagenic treatment- increase rate of mutation

Mutation frequency= mutant cells present in a population

Mutagens-increase the mutation rate

-Chemical

-Physical

-Biological agents


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Mutagenesis1.Chemical mutagens-base analogs-substitutes look alike molecule

-alkylating agentseg. Nitrosoguanidine-introduce changes in nonreplicating DNA

-intercalating agents-insert between two

DNA base pairseg. Ethidium bromide


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2. Radiationa. nonionizing-UV (260 nm) absorbed by nucleotide bases resulting in production ofpyrimidine dimers (two adjacent pyrimidine bases become covalently joined)

b. ionizingshort wavelengths such as X-rays, cosmic rays and gamma rays-substances ionize-free radicals (hydroxyl radical) that react with DNA


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3.Biological and site-directed mutagenesis

a. Transposon mutagenesis

b. Mutations that arise from DNA repair-SOS regulatory system

-some repair occurs without a template-errors


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Transposable elements-transposition- the process by which a gene moves from one place to another in the genome (10-

5 - 10-7/ generation)1. transposase-recognizes, cuts, and ligates DNA2. short inverted terminal repeats at the end of the DNA

(20 bp- 1000bp)

A-insertion sequences (1000 bp)

- IS1, IS2.-found in plasmid, chromsomal, bacteriophage DNA

B-transposons- are insertion sequences with additional genes


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Target sequence is duplicated

-transposons can be random or prefer hot spots


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Ames Test


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Ames Test-bacteria as initial screen -hisG mutant-reversion mutation restores gene

-modify by treating potential mutagen with rat

liver extract

-inexpensive preliminary screen to weed outmutagenic chemicals

Transgenic mice

-mice with lacZ gene inserted into chromosomes-distributed throughout mouse-can find out if certain organs convert harmlessprecursor chemicals into mutagens

Salmonella - His- auxotroph

control

test

Disk with mutagen


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DNA Repair

-type of mutation-extent of damage

Error proof repair-rely on one of the two strands remainingundamaged

Eg. Base excision repair- snips damaged

bases from DNA

Eg. Recombinational repairone daughter strand is undamaged


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Error-Prone DNA repair-SOS response

-a coordinated cellular response to DNA damage

-in order to save the cell, can introduce mutations into severelydamaged DNA


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Gene regulation

How does the cell control all the different reactions involved in a

single cycle of cell growth?

How do microorganisms respond to changes in their environment?


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DNA sequence control-program the rearrangement of DNA so as to activate or disable a particulargene

Phase variation-DNA rearrangement to turn on and off expression of certain cell surfaceproteins

Fig. 9-1


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Transcription

No control

mRNAA

mRNAB

GeneA

Gene B

Translation

EnzymeA

Substrate

Product

Control of enzyme activity No product

Translational control No protein synthesis

Enzyme B

mRNA

C

Transcriptional control No mRNA synthesis

Gene C

Gene D

Fast

Slow

1 R l ti f ti it


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1. Regulation of enzyme activity


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Allosteric inhibition

1. Substrate binding site2. Allosteric site-inhibitor binding site

Covalent modificationEg. adenylation (the addition of AMP)


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Transcriptional control

Induction and repression1. Synthesis of enzymes involved in catabolic pathways can be inducible

-the initial substrate of the pathway (or some derivative of it) isusually the inducer

2. Synthesis of enzymes involved in anabolic pathways is repressible-end product of the pathway usually acts as a corepressor

Regulatory proteins- regulators bind specific small MW ligands to determine their concentrations- bind to specific DNA regulatory sequences


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1. Derepression of Lac Operon- catabolic

-genes that code for the enzymes needed for lactose catabolism

lactose glucose + galactose

Three Structural Genes-three enzymes in the lac catabolic pathway

LacY, LacZ, LacA,

Promoter- DNA segment where RNA polymerase binds and startstranscription

Operator- DNA segment found between the promoter and structuralgenes where the regulatory protein binds

Synthesis of the enzyme only when the substrate is present


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Synthesis of the enzyme only when the substrate is present

induction increases the amount of mRNA encoding the enzymes

Allolactose

-galactosidase

LacI

lacO


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Lac operon

-expressed at low level

-lactose transported into cell in low amounts-LacZ at low levels changes lactose into allolactose

-100X increase in expression of lac operon once lactose added to medium

2 Repression of arginine synthesis


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2. Repression of arginine synthesis(Anabolic pathway)

Total protein synthesis is

unchanged

repression decreases the amount of mRNA encoding the enzymes

Positive control


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-regulator protein promotes the binding of RNA polymerase andincreases mRNA synthesis

Eg.Maltose catabolism

Note: maltose catabolizing enzymes are spread out in various operons all

controlled by the activator protein-regulon

Activator sequence


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Two-Component Phosphorelay SystemsA signal transduction system that uses transfer of phosphoryl groups tocontrol gene transcription and protein activity

-sensor kinase protein- changes in conformation

- autophosphorylates at His residueeg. PhoQ in Salmonella senses magnesium

-response regulator- transphosphorylation at Glu orAsp residue

-phosphatase

Signal transduction and two component systems


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Environmental signal

Sensor kinase

Cytoplasmicmembrane

Response regulator

Transcription blockedDNA

Structural genesOperatorPromoter

RNApolymerase

Signal transduction and two component systems


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Global regulatory systems

-systems that affect many genes and pathways simultaneously, allowingfor both independent regulation of operons as well as cooperation ofoperons

a. a single regulator protein (repressor or activator) to regulate severaloperons

b. different sigma factors

c. nonprotein regulators-small regulatory mRNAs

Regulon-a collection of genes or operons controlled by a commonregulatory protein

R ul ti n b si m f ct rs


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Regulation by sigma factors

Bacteria produce a number of different sigma factors; each enables RNApolymerase to recognize and bind to specific promoters

Alteration of the sigma factors available to RNA polymerase changes geneexpression

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Bio120 LecPPTS Week 10 11

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