microbio chapters 11/12

genome

the genetic material that defines the organism.

Each organism has a unique DNA sequence for that species in its…

genome

the flow of information

1. expression

2. recombination

3. replication

expression

Genetic information is used within a cell to produce the proteins needed for the cell to function.

recombination

Genetic information can be transferred between cells of the same generation.

replication

Genetic information can be transferred between generations of cells.

3 phases of dna synthesis

1. initiation

2. elongation

3. termination

initiation

•involves the unwinding of the helix, priming, and loading of the DNA polymerase enzyme complex.

Elongation

the sequential extension of DNA by adding DNA nucleotide triphosphates (dNTPs) with release of pyrophosphate, followed by proofreading

Termination

which the DNA helix is completely duplicated, and replication stops.

dna polymerase I

exonuclease activity removes RNA primer and replaces it with newly synthesized DNA

DNA polymerase III

main enzyme that adds nucleotides in the 5’ to 3’ direction

helicase

opens the dna helix by breaking hydrogen bonds between the nitrogenous bases

ligase

seals the gaps between the Okazaki fragments on the lagging strand to create one contrinous DNA strand

primase

synthesizes RNA primers needed to start replication

single stranded binding proteins

bind to single stranded dna to prevent hydrogen binding between DNA strands, reforming double stranded DNA

sliding clamp

helps hold. DNA poly III in place when nucleotides are being added

topoisomerase II

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 DNA

topoisomerase IV

introduces single stranded break into concatenated chromosomes to release them from each other and then reseals DNA

what are the stop codons

UAA

UAG

UGA

•Transfer RNAs (tRNA)

•Exist in the cytoplasm

•60 to 90 types

•Bind to specific codon on the mRNA template and add the specific amino acid to the polypeptide chain

translation phases

1. initiation

2. elongation

3. termination

initiation translation

translational complex forms and tRNA brings first amino acid in polypeptide chain to bind to start codon on mRNA

elongation translation

tRNA bring amino acids one by one to add to polypeptide chain

termination translation

release factor recognizes stop codon, translational complex dissociates, and completed polypeptide is released

Point mutation/base substitution

when a single nucleotide is changed in a DNA sequence.

1, silent mutation

2. missense mutation

3. nonsense mutation

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.

mutations causes

•A “mistake” by DNA polymerase that fails to be repaired

1. physical

2. chemical

physical agents that cause mutations

cosmic rays

  X-rays

  UV radiation – cause pyrimidine dimers

chemical agents that cause mutations

reactive oxygen molecules (H₂O₂)

  superoxide radicals (•O₂^–)  

  acridine orange (intercalating agent)

  certain biological processes (mutator strains)

types of DNA repair

1. base excision repair

2. methyl mismatch repair

3. SOS repair

4. 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

1. changing the DNA sequence

2. control of transcription

3. translational control

4. 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. Ex: activate, deactivate or degrade the protein

Some genes respond to changes inside the cell; others respond to outside influences. How do cells assign these tasks?

1.Sensing the intracellular environment

2.Global regulators

3.Sensing the extracellular environment

sensing the intraceullar environment

Different regulatory proteins bind to specific compounds to determine the compound’s concentration.

  Example: dtx, the diphtheria toxin gene

2.Global regulators

  Proteins that affect the expression of many different genes are called global regulators.

EX: cAMP receptor protein (CRP) of E. coli and related species.

3.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 senses magnesium and pH outside the cell.

Series of two-component protein phosphorylation relay systems.

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, transductionand conjugation.  Once the genetic information is transferred from the donor, it can enter the genome of the recipient by recombination.

plasmids

Mostly circular, double-stranded, extrachromosomal DNA

Self replicating by the same mechanisms as any other DNA

Most of plasmids have been identified due to having some function they allow the bacterium to survive.

transposable elements/transposons

Genes that can move from one chromosome to another

•Unlike plasmids, transposable elements cannot replicate outside a larger DNA molecule.

•All transposable elements include a transposasegene whose enzyme product moves the element from one DNA molecule into another.

transformation

•Importing free DNA from the environment into bacterial cells.

•Transformasome.

specific protein complexes that carry out transformation

•Streptococcus, Bacillus, Haemophilus, Neisseria = naturally ….

naturally competent

•E. coli, Salmonella = can be manipulated to be made …

artificially competent

Electroporation

•A brief electrical pulse “shoots” DNA across the membrane.

transformation process

1. competence factor is synthesized and exported

2. as cell numbers rise, external CF level increases and activates sensor kinase

3. sensor kinase transfers a signal (phosphatase) to a transcriptional activator that stimulates transcription of the transformasome genes

4. transformasome binds extraceullar dna. one strand is transported, one strand is degraded.

Transduction

Gene transfer is mediated by a bacteriophage (bacterial virus) vector.

bacteriophage composition

Composed of a core nucleic acid covered by a protein coat

Why is transduction significant?

1.Transfer genetic material from one bacterial cell to another and alters the genetic characteristics of the recipient cell

2.Incorporation of phage DNA into a bacterial chromosome demonstrates a close evolutionary relationship between the prophage and the host bacterial cell

3.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.

4.The viruses bring along genes from their previous host (or hosts) 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

5.It provides a way to study gene linkage to do chromosome mapping

conjugation

Gene transfer requires contact between donor and recipient cells

Larger quantities of DNA are transferred compared to Transduction or Transformation

typical conjugation

1. pilus of donor cell attaches to recipient cell, pilus contracts, drawing cells together to make contact with one antoher

2. one strand of F plasmid DNA transfers from donor cell to recipient cell

3. donor synthesizes complementary strand to restore plasmid. recipient synthesizes complementary strand to become F + cell with pilus

Why is conjugation significant?

1.Contributes to genetic variation

2.Increases genetic diversity (comparing to other mechanisms) - larger amounts of DNA are transferred

3.May represent an evolutionary stage between asexual processes and the actual fusion of whole cells (the gametes)

4.Plasmids are self transmissible and can sometimes be promiscuous

5.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 contain the plasmid to mate with them.

restriction endonuclease.

specific enzymes that can cut DNA plasmids that have

certain DNA sequences

palindrome

Usually the restriction sequence is a “palindrome,” in which the sequence of base pairs reads the same forward and back.

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.

f plasmids

direct synthesis of protein towards the pili

resistance plasmids

carry genes that provide resistance to antimicrobials (chloramphenicol, arsenic, etc)

virulence plasmids

cause disease signs and symptoms

Tumor inducing plasmids –

cause tumors in plants

Genes for catabolic enzymes

not essential for growth

Bacteriocinogen plasmid

Direct synthesis of bacteriocins (bacteria killing)

operon

cluster of genes under the control of one promoter, mostly found in prokaryotes.

lac operon

If lactose is present, it binds to the repressor, which falls off the DNA — now RNA polymerase can do its job.

If no lactose, the repressor stays on the operator and blocks transcription.

This is inducible gene expression — turned on only when needed.

Promoter

where RNA polymerase binds to start transcription.

Operator

on/off switch controlled by a repressor protein

Structural genes:

code for proteins to digest lactose.

inducible gene expression

turned on only when needed

Repression:

Genes are turned off when not needed (ex: tryptophan operon shuts down when there's enough tryptophan).

Induction

Genes are turned on in response to a stimulus (like lactose in the lac operon).

Intracellular:

regulatory proteins inside the cell bind to molecules and influence gene expression.

• Example: diphtheria toxin gene is only active under certain internal conditions.

Extracellular (Two-Component Systems):

• Sensor kinase (in the membrane) detects an external signal.

• Response regulator (in the cytoplasm) changes gene expression.

Ex: Salmonella uses PhoQ to sense low magnesium outside and responds by altering its genes.

Unlike plasmids, transposons cannot…

replicate independently

Specialized transduction

Only certain genes are transferred

significance of specialized transduction

• Transfers traits like toxin genes or antibiotic resistance.

• Suggests close virus-host evolutionary relationships.

• Used to study gene mapping.

F (fertility) plasmid forms?

sex pillus

Hfr cells (High-frequency recombination):

• F plasmid integrates into the chromosome.

• Can transfer part of the host genome, not just plasmid.

soem gram negative bacteria dont use pilli they use…

They use pheromone-like peptides to initiate mating behavior.

What is gel electrophoresis used for?

To separate DNA fragments by size using an electric field.

How does gel electrophoresis work?

DNA (negatively charged) moves through a gel toward the positive end. Smaller fragments move faster.

What can gel electrophoresis tell us?

The size of DNA fragments and whether a sample has a certain gene or mutation.

how is recombinant DNA made?

Using restriction enzymes to cut DNA and ligase to join different DNA pieces.

What are plasmids used for in recombinant DNA?

As vectors to carry and replicate foreign DNA in host cells (like E. coli).

What is the purpose of gene fusion?

To monitor gene expression or put a gene under a different promoter.

What is PCR used for?

To make millions of copies of a specific DNA segment.

What are the three main steps of PCR?

1. denaturation: Heat DNA to separate strands

1. Annealing: Cool to let primers bind

2. Extension: DNA polymerase adds nucleotides

What enzyme is used in PCR?

Taq polymerase – a heat-stable enzyme from Thermus aquaticus.

What is a primer in PCR?

A short single-stranded DNA piece that starts the replication process.