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© 2016 Pearson Education, Inc.
The Regulation of Bacterial Gene Expression
• Constitutive genes are expressed at a fixed rate • Other genes are expressed only as needed
• Inducible genes • Repressible genes • Catabolite repression
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Pre-transcriptional Control
• Repression inhibits gene expression and decreases enzyme synthesis • Mediated by repressors, proteins that block
transcription • Default position of a repressible gene is on
• Induction turns on gene expression • Initiated by an inducer • Default position of an inducible gene is off
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The Operon Model of Gene Expression
• Promoter: segment of DNA where RNA polymerase initiates transcription of structural genes
• Operator: segment of DNA that controls transcription of structural genes
• Operon: set of operator and promoter sites and the structural genes they control
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The Operon Model of Gene Expression
• In an inducible operon, structural genes are not transcribed unless an inducer is present • In the absence of lactose, the repressor binds to the
operator, preventing transcription • In the presence of lactose, lactose (inducer) binds to the
repressor; the repressor cannot bind to the operator and transcription occurs
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Figure 8.12 An inducible operon (1 of 3).
Control region Structural genes
Operon
I P O Z Y A
DNA
Regulatory gene
Promoter Operator
Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I)
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Figure 8.12 An inducible operon (2 of 3).
RNA polymerase
I P Z Y A
Transcription
Translation
Repressor mRNA
Active repressor protein
Repressor active, operon off. The repressor protein binds with the operator, preventing transcription from the operon.
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Allolactose (inducer)
I P O Z Y A
Transcription
Translation
Transacetylase Permease
β-Galactosidase
Inactive repressor protein
Repressor inactive, operon on. When the inducer allolactose binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately resulting in the production of the enzymes needed for lactose catabolism.
Operon mRNA
Figure 8.12 An inducible operon (3 of 3).
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The Operon Model of Gene Expression
• In repressible operons, structural genes are transcribed until they are turned off • Excess tryptophan is a corepressor that binds and
activates the repressor to bind to the operator, stopping tryptophan synthesis
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Figure 8.13 A repressible operon (1 of 3).
Control region Structural genes
Operon
I P O E C A
DNA
Regulatory gene
Promoter Operator
Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I)
D B
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RNA polymerase
I P O E D C B A
Transcription
Repressor mRNA
Translation
Inactive repressor protein
Polypeptides comprising the enzymes for tryptophan synthesis
Operon mRNA
Repressor inactive, operon on. The repressor is inactive, and transcription and translation proceed, leading to the synthesis of tryptophan.
Figure 8.13 A repressible operon (2 of 3).
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Figure 8.13 A repressible operon (3 of 3).
I P E D C B A
Active repressor protein
Tryptophan (corepressor)
Repressor active, operon off. When the corepressor tryptophan binds to the repressor protein, the activated repressor binds with the operator, preventing transcription from the operon.
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Check Your Understanding
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Positive Regulation
• Catabolite repression inhibits cells from using carbon sources other than glucose
• Cyclic AMP (cAMP) builds up in a cell when glucose is not available
• cAMP binds to the lac promoter, initiating transcription and allowing the cell to use lactose
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Figure 8.14 The growth rate of E. coli on glucose and lactose. Bacteria growing on glucose as the sole carbon source grow faster than on lactose.
Bacteria growing in a medium containing glucose and lactose first consume the glucose and then, after a short lag time, the lactose. During the lag time, intra- cellular cAMP increases, the lac operon is transcribed, more lactose is transported into the cell, and β-galacto- sidase is synthesized to break down lactose.
Glucose
Lactose
All glucose consumed
Glucose used Lag
time
Lactose used
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Figure 8.15 Positive regulation of the lac operon. Promoter
lacZ lacl DNA
Operator RNA polymerase can bind and transcribe
cAMP
Inactive CAP
Active CAP Inactive lac
repressor
Lactose present, glucose scarce (cAMP level high). If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for lactose digestion.
CAP-binding site
CAP-binding site
DNA lacl
Promoter lacZ
Operator RNA polymerase can't bind
Inactive CAP
Inactive lac repressor
Lactose present, glucose present (cAMP level low). When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription.
cAMP – cyclic AMP
CAP – catabolic activator protein
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Epigenetic Control
• Methylating nucleotides turns genes off • Methylated (off) genes can be passed to offspring
cells • Not permanent
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Post-Transcriptional Control
• microRNAs (miRNAs) base pair with mRNA to make it double-stranded
• Double-stranded RNA is enzymatically destroyed, preventing production of a protein
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Figure 8.16 MicroRNAs control a wide range of activities in cells. DNA
Transcription of miRNA occurs.
miRNA miRNA binds to target mRNA that has at least six complementary bases.
mRNA
mRNA is degraded.
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Changes in Genetic Material
• Mutation: a permanent change in the base sequence of DNA
• Mutations may be neutral, beneficial, or harmful • Mutagens: agents that cause mutations • Spontaneous mutations: occur in the absence of
a mutagen
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Types of Mutations
• Base substitution (point mutation) • Change in one base in DNA
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Figure 8.17 Base substitutions.
During DNA replication, a thymine is incorporated opposite guanine by mistake.
Daughter DNA
mRNA
Amino acids Cysteine Tyrosine Cysteine Cysteine
Translation
Transcription
Replication
Replication
Daughter DNA
Daughter DNA
Parental DNA
If not corrected, in the next round of replication, adenine pairs with the new thymine, yielding an AT pair in place of the original GC pair.
When mRNA is transcribed from the DNA containing this substitution, a codon is produced that, during translation, encodes a different amino acid: tyrosine instead of cysteine.
Granddaughter DNA
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Types of Mutations
• Missense mutation • Base substitution results in change in an amino acid
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Figure 8.18a-b Types of mutations and their effects on the amino acid sequences of proteins.
DNA (template strand)
Transcription
Translation
mRNA
Amino acid sequence
Met
Lys Phe Gly Stop
Normal DNA molecule
Stop Lys Phe
DNA (template strand)
Amino acid sequence
Missense mutation
Met
mRNA
Ser
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Types of Mutations
• Nonsense mutation • Base substitution results in a nonsense (stop) codon
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Figure 8.18a-c Types of mutations and their effects on the amino acid sequences of proteins. DNA (template strand)
Transcription
Translation
mRNA
Amino acid sequence Lys Phe Gly Stop
Normal DNA molecule
Met
Stop Met
Nonsense mutation
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Types of Mutations
• Frameshift mutation • Insertion or deletion of one or more nucleotide pairs • Shifts the translational "reading frame"
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Figure 8.18a-d Types of mutations and their effects on the amino acid sequences of proteins. DNA (template strand)
Transcription
Translation
mRNA
Amino acid sequence Lys Phe Gly Stop
Normal DNA molecule
Met
Frameshift mutation
Met Lys Leu Ala
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Chemical Mutagens
• Nitrous acid: causes adenine to bind with cytosine instead of thymine
• Nucleoside analog: incorporates into DNA in place of a normal base; causes mistakes in base pairing
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Figure 8.19a Oxidation of nucleotides makes a mutagen.
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Figure 8.19b Oxidation of nucleotides makes a mutagen.
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Figure 8.20 Nucleoside analogs and the nitrogenous bases they replace.
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Radiation
• Ionizing radiation (X rays and gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone
• UV radiation causes thymine dimers
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Radiation
• Photolyases separate thymine dimers • Nucleotide excision repair: Enzymes cut out
incorrect bases and fill in correct bases
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Figure 8.21 The creation and repair of a thymine dimer caused by ultraviolet light. Ultraviolet light
Exposure to ultraviolet light causes adjacent thymines to become cross-linked, forming a thymine dimer and disrupting their normal base pairing. Thymine dimer
An endonuclease cuts the DNA, and an exonuclease removes the damaged DNA.
New DNA DNA polymerase fills the gap by synthesizing new DNA, using the intact strand as a template.
DNA ligase seals the remaining gap by joining the old and new DNA.
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The Frequency of Mutation
• Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes
• Mutagens increase the mutation rate to 10–5 or 10–3 per replicated gene
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Identifying Mutants
• Positive (direct) selection detects mutant cells because they grow or appear different than unmutated cells
• Negative (indirect) selection detects mutant cells that cannot grow or perform a certain function
• Auxtotroph: mutant that has a nutritional requirement absent in the parent • Use of replica plating
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Figure 8.22 Replica plating.
Handle
Sterile velvet is pressed on the grown colonies on the master plate.
Velvet surface (sterilized)
Master plate with medium containing histidine
Plates are incubated.
Petri plate with medium containing histidine
Auxotrophic mutant
Growth on plates is compared. A colony that grows on the medium with histidine but could not grow on the medium without histidine is auxotrophic (histidine-requiring mutant).
Colony missing
Petri plate with medium lacking histidine
Cells from each colony are transferred from the velvet to new plates.
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Identifying Chemical Carcinogens
• The Ames test exposes mutant bacteria to mutagenic substances to measure the rate of reversal of the mutation • Indicates degree to which a substance is mutagenic
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Figure 8.23 The Ames reverse gene mutation test.
Experimental sample
Control (no suspected mutagen added)
Rat liver extract
Suspected mutagen
Cultures of histidine-dependent Salmonella Media lacking histidine
Colonies of revertant bacteria
Rat liver extract
Experimental plate
Incubation
Incubation
Two cultures are pre- pared of Salmonella bacteria that have lost the ability to synthesize histidine (histidine- dependent).
The suspected mutagen is added to the experimental sample only; rat liver extract (an activator) is added to both samples.
Each sample is poured onto a plate of medium lacking histidine. The plates are then incubated at 37°C for two days. Only bacteria whose histidine-dependent phenotype has mutated back (reverted) to histidine- synthesizing will grow into colonies.
The numbers of colonies on the experimental and control plates are compared. The control plate may show a few spontaneous histidine-synthesizing revertants. The test plates will show an increase in the number of histidine-synthesizing revertants if the test chemical is indeed a mutagen and potential carcinogen. The higher the concentration of mutagen used, the more revertant colonies will result.
Control plate
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Genetic Transfer and Recombination
• Genetic recombination: exchange of genes between two DNA molecules; creates genetic diversity
• Crossing over: Two chromosomes break and rejoin, resulting in the insertion of foreign DNA into the chromosome
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Figure 8.24 Genetic recombination by crossing over.
DNA from one cell aligns with DNA in the recipient cell. Notice that there is a nick in the donor DNA.
DNA from the donor aligns with complementary base pairs in the recipient's chromosome. This can involve thousands of base pairs.
RecA protein catalyzes the joining of the two strands.
The result is that the recipient's chromosome contains new DNA. Complementary base pairs between the two strands will be resolved by DNA polymerase and ligase. The donor DNA will be destroyed. The recipient may now have one or more new genes.
RecA protein
Recipient chromosome
Donor DNA
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Genetic Transfer and Recombination
• Vertical gene transfer: transfer of genes from an organism to its offspring
• Horizontal gene transfer: transfer of genes between cells of the same generation
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Transformation in Bacteria
• Transformation: genes transferred from one bacterium to another as "naked" DNA
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Figure 8.25 Griffith's experiment demonstrating genetic transformation.
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Figure 8.26 The mechanism of genetic transformation in bacteria.
a b c d
DNA fragments from donor cells
Recipient cell
A D
B C
Chromosomal DNA
Recipient cell takes up donor DNA.
Donor DNA aligns with complementary bases.
Recombination occurs between donor DNA and recipient DNA.
A D
B C
A D
B C
Degraded unrecombined DNA
Genetically transformed cell
a b c d
b c d
B C
D a
5' 3'
5' 3'
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Conjugation in Bacteria
• Conjugation: plasmids transferred from one bacterium to another
• Requires cell-to-cell contact via sex pili
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Figure 8.27 Bacterial conjugation.
Sex pilus
F– cell
Mating bridge
F+ cell
Sex pilus Mating bridge
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Conjugation in Bacteria
• Donor cells carry the plasmid (F factor) and are called F+ cells
• Hfr cells contain the F factor on the chromosome
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Figure 8.28a Conjugation in E. coli.
RECOMBINATION
Bacterial chromosome
Mating bridge
Replication and transfer of F factor
F factor
F+ cell F– cell When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F–), the F– cell is converted to an F+ cell.
F+ cell F+ cell
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Figure 8.28b Conjugation in E. coli.
RECOMBINATION
Recombination between F factor and chromosome, occurring at a specific site on each
Insertion of F factor into chromosome
Integrated F factor
F+ cell When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a high frequency of recombination (Hfr) cell.
Hfr cell
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Figure 8.28c Conjugation in E. coli.
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Conjugation in Bacteria
• Conjugation can be used to map the location of genes on a chromosome
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Figure 8.29 A genetic map of the chromosome of E. coli.
bp1
3480 kbp
2320 kbp
1160 kbp
Amino acid metabolism
DNA replication and repair
Lipid metabolism
Carbohydrate metabolism
Membrane synthesis
80
90 0
10
20
30
40 50
60
70
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Transduction in Bacteria
• DNA is transferred from a donor cell to a recipient via a bacteriophage
• Generalized transduction: Random bacterial DNA is packaged inside a phage and transferred to a recipient cell
• Specialized transduction: Specific bacterial genes are packaged inside a phage and transferred to a recipient cell
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Figure 8.30 Transduction by a bacteriophage. The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.RECOMBINATION
Phage protein coat Phage DNA
Bacterial chromosome
A phage infects the donor bacterial cell.
Phage DNA and proteins are made, and the bacterial chromosome is broken into pieces.
Occasionally during phage assembly, pieces of bacterial DNA are pack- aged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA.
Phage DNA Bacterial DNA A phage carrying
bacterial DNA infects a new host cell, the recipient cell. Recipient
cell
Donor bacterial DNA
Recipient bacterial DNA
Recombinant cell reproduces normally
Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.
Many cell divisions
Donor cell
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Plasmids
• Plasmids are self-replicating circular pieces of DNA
• 1 to 5% the size of a bacterial chromosome • Often code for proteins that enhance the
pathogenicity of a bacterium
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Figure 8.31 R factor, a type of plasmid. Origin of
replication
Pilus and conjugation
proteins
Origin of transfer
Tetracycline resistance
Chloram- phenicol resistance
Streptomycin resistance
Sulfonamide resistance
Mercury resistance
RTF
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Plasmids
• Conjugative plasmid: carries genes for sex pili and transfer of the plasmid
• Dissimilation plasmids: encode enzymes for the catabolism of unusual compounds
• Resistance factors (R factors): encode antibiotic resistance
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Transposons
• Transposons are segments of DNA that can move from one region of DNA to another
• Contain insertion sequences (IS) that code for transposase that cuts and reseals DNA
• Complex transposons carry other genes (e.g, in antibiotic resistance)
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Figure 8.32a Transposons and insertion.
IS1
Inverted repeat Inverted repeat An insertion sequence (IS), the simplest transposon, contains a gene for transposase, the enzyme that catalyzes transposition. The tranposase gene is bounded at each end by inverted repeat sequences that function as recognition sites for the transposon. IS1 is one example of an insertion sequence, shown here with simplified IR sequences.
Transposase gene
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Figure 8.32b-c Transposons and insertion.
Transposase gene
Transposase cuts DNA, leaving sticky ends.
Tn5
IS1
Complex transposons carry other genetic material in addition to transposase genes. The example shown here, Tn5, carries the gene for kanamycin resistance and has complete copies of the insertion sequence IS1 at each end.
Kanamycin resistance
IS1 IS1
IS1 Sticky ends of transposon and target DNA anneal.
Insertion of the transposon Tn5 into R100 plasmid
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Genes and Evolution
• Mutations and recombination create cell diversity • Diversity is the raw material for evolution • Natural selection acts on populations of organisms
to ensure the survival of organisms fit for a particular environment
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