ch 11 & 12: mechanisms of microbial genetics Flashcards
genome
the genetic material that defines the organism
- each organism has a unique DNA sequence for that species in its genome
the flow of genetic information
genetic information can be transferred in 3 ways:
1. expression
2. recombination
3. replication
the flow of genetic information: expression
genetic information is used within a cell to produce the proteins needed for the cell to function
- cell undergoes transcription and translation
- cell metabolizes and grows
the flow of genetic information: recombination
genetic information can be transferred between cells of the same generation
- new combination of genes in DNA in the recombinant cell
the flow of genetic information: replication
genetic information can be transferred between generations of cells
- forms two daughter cells
the central dogma
states that DNA encodes messenger RNA, which, in turn, encodes proteins
DNA———>RNA———->protein
(transcription) (translation)
^referred to as gene expression^
exceptions to the central dogma
- reverse transcriptase
- prions
phenotype
the product of the array of proteins being produced by the cell at a given time
- it is influenced by the cell’s genotype
- as well as interaction with the cell’s environment
Genotype leading to different phenotypes
genotype–> environmental condition A/B —> phenotype A/B
the phenotype is the product of the proteins being produced by the cell
- which is influenced by the cell’s genotype as well as interactions with the cell’s environment
ex: Serratia marcscens - different temperature yields different phenotypes
nucleic acids
long chains composed of nucleotides
nucleotide
3 components:
- 5 carbon sugar
- phosphate group
- nitrogenous base (nucleobase)
Deoxyribonucleic Acid (DNA)
encodes genetic information in genes
Ribonucleic Acid (RNA)
transfer of information from gene to protein
DNA nucleotide
deoxyribosenucleotide
- each is made up of: deoxyribose (sugar), a phosphate group, and a nitrogenous base (i.e. A, T, G, C)
- the 5 carbons within the deoxyribose carbon ring are designated as 1’, 2’, 3’, 4’, 5’
purines
has 2 carbon rings
- adenine
- guanine
pyrimidines
contains one carbon ring
- cytosine
- thymine
- uracil
DNA structure
- double stranded helix
- complementary base pairing
- Adenine (purine) and thymine (pyrimidine) pair by 2 hydrogen bonds
- Guanine (purine) and cytosine (pyrimidine) pair by 3 hydrogen bonds
major and minor grooves form when the two strands twist around each other
DNA double helix
free phosphate group at the 5’ carbon end and a free hydroxyl group at the 3’ carbon end
phosphodiester bonding between nucleotides forms the sugar-phosphate backbone
RNA structure
ribonucleotides contain the pentose sugar ribose instead of deoxyribose found in deoxyribonucleotides
- RNA containes uracil instead of thymine
single stranded structure
- RNA can fold upon itself
- folds stabilized by short areas of complementary base pairing within the molecule → 3D structure
stages of DNA replication
1. initiation
-involves unwinding of the helix, priming, and loading of the DNA polymerase enzyme complex
2. elongation
-the sequential extension of DNA by adding deoxyribonucleoside triphosphates (dNTPs) with release of pyrophosphate, followed by proofreading
3. termination
-the DNA duplication is complete and replication stops
- bacterial topoisomerase IV used
stages of DNA replication: initiation
involves unwinding of the helix, priming, and loading of the DNA polymerase enzyme complex
stages of DNA replication: elongation
the sequential extension of DNA by adding deoxyribonucleoside triphosphates (dNTPs) with release of pyrophosphate (PPi)
- followed by proofreading
flow of genetic information (DNA)
3 types:
- expression
- genetic information is used within a cell to produce the proteins needed for the cell to function
- at this stage, transcription and translation occurs
- within the cell - recombination
- genetic information can be transferred between cells of the same generation
- recombinant cell will have new combination of genes
- within generations - replication
- genetic information can be transferred between generations of cells
- parent cell to daughter cells (between generations)
what are the enzymes used in bacterial DNA replication?
- DNA polymerase I
- DNA polymerase III
- helicase
- ligase
- primase
- single-stranded binding proteins
- sliding clamp
- topoisomerase II (DNA gyrase)
- topoisomerase IV
DNA replication: DNA polymerase I
exonuclease activity removes RNA primer and replaces it with newly synthesized DNA
DNA replication: DNA polymerase III
main enzyme that adds nucleotide in the 5’ to 3’ direction
DNA replication: helicase
opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
DNA replication: ligase
seals the gaps between the Okazaki fragments on the lagging strand to create one continuous DNA strand
DNA replication: primase
synthesizes RNA primers needed to start replication
DNA replication: single-stranded binding proteins
bind to single-stranded DNA to prevent hydrogen bonding between DNA strands, reforming double-stranded DNA
DNA replication: sliding clamp
helps hold DNA polymerase III in place when nucleotides are being added
DNA replication: topoisomerase II (DNA gyrase)
relaxes supercoiled chromosome to make DNA more accessible for the initiation of replication
- helps relieve the stress on DNA when unwinding, by causing breaks and then resealing the DNA
DNA replication: toposiomerase IV
introduces single-stranded break into concatenated chromosomes to release them from each other
- and then after will reseal DNA
Okazaki fragments
short sections of DNA formed at the time of discontinuous synthesis of the lagging strand during replication of DNA
- essential as it allows for the synthesis of both the daughter strands required for cell division
semiconservative DNA replication
each daughter double helix obtains one old and one new strand
- each strand of the double helix DNA serves as a template for synthesis of a new strand
Oirigin of replication
where replication begins
Point mutation/base substitution
when a single nucleotide is changed in a DNA sequence
consist of:
* silent mutation
* missense mutation
* nonsense mutation
Silent mutation
change has no effect on protein structure
missense mutation
a base-pair substituion that results in a codon that codes for different amino acid
nonsense mutation
replaces an amino acid with a stop codon
Insertion/deletion
involves the addition or subtraction of one or more nucleotides
- frameshift mutation
Inversion
occurs when a fragment of DNA is flipped in orientation in relation to the DNA on the other side
what causes mutations
- a “mistake” by DNA polymerase that fails to be repaired
- physical agents (i.e. cosmic rays, x-rays, UV radiation)
- chemical agents
Types of DNA repair
- base excision repair
- methyl mismatch repair
- SOS repair
- DNA recombination
Base excision repair
recognizes a specific damaged base and removes it from the DNA backbone
methyl mismatch repair
requires recognition of the methylation pattern in DNA bases
SOS (save our ship) repair
coordinated cellular response to damage that can introduce mutations in order to save the cell
DNA recombination
the process of “crossing over” and exchange of two DNA helices
Levels of gene regulation
- changing the DNA sequence
- control of transcription
- translational control
- post-translational control
changing the DNA sequence
some microbes change the DNA sequence to activate or disable a particular gene
ex: phase variation
control of transcription
transcription can be regulated by protein repressors, activators, and alternative sigma factors
translational control
control of transcription initiation sequences that recognize specific repressor proteins
post-translational control
control of proteins that are already made
operon
where structure proteins with related functions are usually encoded together
includes:
- promoter
- operator
- structural genes
Repression
prokaryotic operons are commonly controlled by the binding of repressors to operator regions, which prevents the transcription of the structural genes
repressible operons
tryptophan
- typically contain genes encoding enzymes required for a biosynthetic pathway
- as long as the product of the pathway (trp) continues to be required by the cell, a repressible operon will continue to be expressed
- when the product begins to accumulate, the expression of the operon is repressed
Inducible operons
lac operon
- contain genes encoding enzymes in a pathway involved in the metabolism of a specific substrate like lactose
- these enzymes are only required when that substrate is available, thus expression of the operons is typically induced only in the presence of the substrate
Some genes respond to changes inside the cell; others respond to outside influences. How do cells assign these tasks?
- sensing the intracellular environment
- global regulators
- sensing the extracellular environment
Sensing the intracellular environment
different regulatory proteins bind to specific compounds to determine the compounds concentration
ex: dtx, the diphtheria toxin gene
iron binds to dtxR–> dtxR binds to regulatory region and no toxin expressed
OR
without iron, dtxR comes off DNA and toxin is expressed
Global regulators
proteins that affect the expression of many different genes
ex: cAMP receptor protein (CRP) of E. coli and related species
Sensing the extracellular environment
a common mechanism used by bacteria to sense outside of the cell and transmit that information inside relies on a series of two-component protein phosphorylation relay systems
ex: sensor kinase PhoQ in Salmonella
sensor kinase PhoQ in Salmonella
- sensor kinase detects condition outside the cell
- signal triggers (or prevents) autophosphorylation
- phosphate is transferred to a response regulator in the cytoplasm. Regulator binds DNA and either stimulates or represses the target genes
- a phosphatase removes the phosphate and down regulates the system
Vertical gene transfer
- occurs during reproduction between generations of cells
- exchange of genes between two DNA molecules
- crossing over occurs when two chromosomes break and rejoin
Horizontal gene transfer
the transfer of genes between cells of the same generation
- also called lateral gene transfer - transformation, transduction and conjugation.
- once the genetic information is transferred from the donor, it can enter the genome of the recipient by recombination
Transformation
in transformation, the cell takes up DNA directly from the environment. the DNA may remain separate as a plasmid or be incorporated into the host genome
Transduction
a bacteriophage injects DNA that is a hybrid of viral DNA and DNA from a previously infected bacterial cell
Conjugation
DNA is transferred between cells through a cytoplasmic bridge after a conjugation plus draws the two cells close together
Plasmids
- mostly circular, double-stranded, extrachromosomal DNA
- self replicating by the same mechanisms as any other DNA
- most of the plasmids have been identified due to having some function they allow the bacterium to survive
F plasmids
direct synthesis of proteins towards the pili
Resistance (R) plasmids
carry genes that provide resistance to antimicrobials (chloramphenicol, arsenic etc)
Virulence plasmids
(neurotoxin) cause disease signs and symptoms
Tumor inducing plasmids
cause tumor formation in plants
Genes for catabolic enzymes
not essential for cell growth
Bacteriocinogen plasmid
direct synthesis of bacteriocins (bacteria killing)
Transposable elements or transposons
- genes that can move from one chromosome to another
- exist in virtually all life forms
- unlike plasmids, transposable elements cannot replicate outside a larger DNA molecule
- all transposable elements include a transposase gene whose enzyme product moves the element from one DNA molecule into another
Transformation
- importing free DNA from the environment into bacterial cells
- natural transformation is a property inherent to many bacterial species and is carried out by specific protein complexes - transformasome
- Streptococcus, Bacillus, Haemophilus, Neisseria = naturally competent
- E. coli, Salmonella = can be manipulated to be made artificially competent
- Electroporation: a brief electrical pulse shoots DNA across the membrane
the process of transformation
- competence factor (CF) is synthesized and exported
- as cell numbers rise, external CF level increases and activates sensor kinase
- the sensor kinase transfers a signal (phosphate) to a transcriptional activator that stimulates transcription of the transformasome gene
- transformasome binds extracellular DNA. One strand is transported; one strand is degraded
Transduction
gene transfer is mediated by a bacteriophage vector
Trans “across” ductio “ to pull”
- originally discovered in Salmonella in 1952
Specialized transduction
- phage DNA integrates into host DNA
- copies of phage DNA include adjacent host DNA (gene D)
- Lysis releases progeny phage whose genome includes gene D
- phage including gene D infects recipient cell
- phage genome integrates with host genome including gene D from donor cell
Why is transduction significant
- transfer genetic material from one bacterial cell to another and alters the genetic characteristics of the recipient cell
- incorporation of phage DNA into a bacterial chromosome demonstrates a close evolutionary relationship between the prophage and host bacterial cell
- the fact that a prophage can remain for long periods of time in a cell suggests a similar mechanism for the viral origin of cancer
- the viruses bring alone genes from their previous host, thus, if this type of virus infects us, the type of DNA incorporated into us might belong to another animal, AKA we might consider ourselves transgenic
- it provides a way to study gene linkage to do chromosome mapping
Typical conjugation steps
- pilus of donor cell attaches to recipient cell. pilus contracts, drawing the cells close together
- one strand of F plasmid DNA transfers from donor cell to recipient cell
- donor synthesizes complementary strand to restore plasmid. recipient synthesizes complementary strand to become F+ cell with pilus
Hfr cell
“Hfr” refers to the high frequency of recombination seen when the recipient F- cells receive genetic information from Hfr cells through conjugation
form when F plasmid integrates into the bacterial chromosome through recombination
Imprecise excision of the F plasmid from the chromosome of an Hfr cell may lead to the production of an F’ plasmid that carries chromosomal DNA adjacent to the integration site
Why is conjugation significant?
- contributes to genetic diversity - larger amounts of DNA are transferred
- may represent an evolutionary stage between asexual processes and the actual fusion of whole cells (the gametes)
- plasmids are self transmissible and can sometimes be promiscuous
- some gram + bacteria have self mating plasmids that do not form a F pili, instead they secrete peptide compounds which simulate nearby bacteria that do not contain the plasmid to mate with them
Plasmids in biotechnology
DNA plasmids have certain DNA sequences that can be cut by a specific enzyme called a restriction endonuclease
* usually the restriction sequence is a “palindrome”, in which the sequence of the base pairs reads the same forward and back
Gel electrophoresis
technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity
- DNA has a negative charge and will be drawn to the positive electrode
- smaller molecules travel faster
Gene fusion
- transposition of genes from one location of the chromosome to another - fusion of two genes together
- depending on the design - a function may be inactivated or a function may be placed under the control of a different regulatory sequence
