Microbial Genetics

Genetics

the study of heredity; includes the study of the genes

gene replication

gene products (proteins, rRNA, tRNA)

traits derived from expressed genes and how they are passed from one generation to the other

Genome

sum of the cells genetic material

consists of all of the chromosomes of a cell and plasmids

genomics

the sequencing and molecular characterization of genomes

genotype

genetic makeup of cell/organism

Genotype

genetic makeup of cell/organism

phenotype

actual traits due to the expression of the genotype (the expression of the genes as a trait)

chromosomes

the physical structure that carries the hereditary information

What are genes made of

made from DNA (which is made of nucelotides) with A, T, C, G

What is the structure of DNA

double-stranded helical DNA, acts as a template to make RNA

has two complementary strands

Prokaryotes chromosomes

they have one circular chromosomes but may also contain a plasmid

eukaryotes chromosomes

have more than one chromosomes that are linear

What is DNA made of

it is made of nucleotides

it has a nitrogenous base + deoxyribose + phosphate

sugar-phosphate backbone

Hydrogen bonds form between A-T and C-G

Dogma of genetic information

dna is turned into RNA into protein

transcription

dna is turned into mRNA

RNA hydrogen bonds

A-U and C-G

Translation

mRNA is used to make protein

Polymers of Nucleotides in DNA

Adenine, thymine, cytosine, and guanine

this forms the double helix associated with proteins, where the strands are held together by hydrogen bonds between AT and CG

antiparallel

What is the backbone of DNA

Deoxyribose-phosphate

DNA replication step 1

the double helix of the parental DNA separates as weak hydrogen bonds between the nucleotides on opposite strands break in response to the action of replication enzymes

DNA replication step 2

hydrogen bonds form between new complementary nucleotides and each strand of the parental template to form new base pairs

DNA replication step 3

Enzymes catalyze the formation of sugar-phosphate bonds between sequential nucleotides on each resulting daughter strand

Overview of dna replication 1

• set of enzymes working in a specific sequence

• process is very accurate

• each parental strand acts as a template for the new daughter strand where 2 daughter strands produced

  • semi-conservative

• all dna is synthesized in a direct manner

  • always from the 5 towards the 3 end of the new strand

• begins at the origin or replication

• bi-directional all the way around → 2 circular dna molecules

DNA replication overview 2

• several enzymes work together in a sequential manner

• dna polymerase adds nucleotides to the growing dna strand

  • in the 5 → 3 direction

  • initiated by an rna primer

  • leading strand is synthesized continuously

  • lagging strand is synthesized discontinuously creating okasaki fragments

  • dna polymerase removes RNA primers; okasaki fragments are joined y the DNA polymerase and dna ligase

1st event at dna replication fork

enzymes unwind the parental double helix

2nd event at dna replication fork

protein stabilize the unwound parental DNA

3rd event at dna replication fork

the leading strand is synthesized continously by dna polymerase

4th event at dna replication fork

the lagging strand is synthesized discontinously primase and rna polymerase synthesizes a short rna primer which is then extended by dna polymerase

5th event at dna replication fork

dna ploymerase digests rna primer and replaces it with dna

6th event at dna replication fork

dna ligase joins the discontinous fragments of the lagging strand

three types of RNA

mRNA- messenger

tRNA- transfer

rRNA- ribosomal

mRNA

“Read” by ribosomes to make protein, carries coding information

tRNA

• Carries individual amino acids to the ribosomes for making the new proteins

rRNA

Ribosomes contain proteins + rRNA

RNA transcription

it is the synthesis of RNA from DNA

nucleotides of rna

a, u, c, g

What is the enzyme of RNA transcription?

DNA dependent RNA polymerase

where does rna transcription start

a site called the promoter

what direction does the enzyme go

it elongates in a 5’ to 3’ direction

where does termination occur in rna transcription

-it occurs as it reaches a terminator codon

What is released at the end of the rna transcription

the enzyme used in transcription is released

1st step of Rna transcription

Initiation of RNA transcription as RNA polymerase binds to the promoter and DNA unwinds at the beginning of a gene

2nd step of RNA transcription

elongation of rna as it is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA

3rd step of RNA transcription

the site of synthesis moves along DNA; DNA that has been transcribed rewinds

4th step of RNA transcription

termination as transcription reaches the terminator

5th step of RNA transcription

RNA and RNA polymerase are released and the DNA helix reforms

Translation of RNA to a Protein

• rna is read in the 5’ to 3’ direction

translation of rna to protein in prokaryotes

transcription and translation is coupled and both occur in the cytoplasm, m-RNA is polycystronic

what language is mRNA in

it is in the form of codons

What do you need for translation of RNA to proteins

ribosomes, t-rna, mrna, amino acids, and translation factors

4 nucleotides of genetic code

A, U, C, and G

used in combinations of 3 gives 64 sets of triplets

1 set of 3 nt = codon

1 start

AUG codes for methionine

3 Stops

UAA, UAG, UGA, non coding

1st step of translation

components needed to begin translation come together

2nd step of translation

on the assembled ribosomes a tRNA carrying the first amino acid is paired with the start codon of the mRNA

the place where this first tRNA sits is called the P site

a tRNA carrying the second amino acid approaches

3rd step of translation

the second codon of the mrna pairs with a trna carrying the second amino acid at the A site

the first amino acid joins to the second by a peptide bond

this attaches the polypeptide to the tRNA in the P site

4th step of translation

the ribosome moves along the mrna until the second tRNA is in the P site

the next codon to be translated is brought into the A site

the first tRNA now occupies the E site

5th step of translation

the second amino acid joins to the third by another peptide bond and the first trna is released from the e site

6th step of translation

the ribosome continues to move along the mRNA and new amino acids are added to the polypeptide

7th step of translation

when the ribosome reaches a stop codon the polypeptide is released

8th step of translation

finally the last tRNA is released and the ribosome comes apart

the released polypeptide forms a new protein

Mutations

permanent, inherited change in the nucleotide sequence of the DNA

changes in the genotype and this change is passed onto the daughter cells

may alter the phenotype if the DNA change causes the codon to code for a different amino acid

spontaneous with no known cause and errors from dna replication

induced caused by a mutagenic agent

Types of mutations

point mutation or base substitution

frameshift mutation

Point mutation or base substitution

missense mutation: change causes a different AA to be used

nonsense mutation: nt changes results in a stop codon

silent mutation: no change occurs in amino acid or protein because the genetic code is degenerate

Frameshift mutation

insertion or deletion of 1 or a few bases

often creates a stop codon

1st step in base substitutions

during dna replication a thymine is incorporated opposite guanine by mistake

2nd step in base substitutions

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

3rdstep in base substitutions

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

Mutagens

physical or chemical factors that cause a change in the dna (a mutation)

2 types: chemical and radiation

Chemical Mutagens

base analogs: similar structure, 2-aminopurine and 5 bromouracil

mitrous acid

alkylating agents

intercalating agents

Radiation Mutagens

ionizing: gamma and xrays

non-ionizing: UV light (sun tanning)

• caused thymine dimers

Repair

photolyases separate thymine dimers

nucleotide excision repair

1st step of repair

exposure to ultraviolet light causes adjacent thymines to become cross-linked forming a thymine dimer and disrupting their normal base pairing

2nd step of repair

An endonuclease cuts the DNA and an exonuclease removes the damaged DNA

3rd step of repair

DNA polymerase fills the gap by synthesizing new DNA using the intact strand as a template

Genetic transfer and recombination

gene transfer is movement of genetic information between organisms

in eukaryotic organisms this can occur during the fertilization of an egg

in prokaryotic organisms this is not an essential part of the life cycle

• when it does occur DNA is transferred from a donor to a recipient cell

combining genes DNA from two different DNA molecules this means genetic recombination

• resulting cells are A RECOMBINATE

Bacteria have 3 ways to transfer DNA

• transformation, transduction, and conjugation

1st step of 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

2nd step of genetic recombination by crossing over

DNA from the donor aligns with complementary base pairs in the recipients chromosome

this can involve thousands of base pairs

3rd step of genetic recombination by crossing over

RecA protein catalyzes the joining of the two strands

4th step of genetic recombination by crossing over

The result is that the recipients 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

Transformation

Naked DNA from cell to cell

susceptible to DNase degradation

Transduction

transfer occurs by viral transfer

bacteriophage

two types of bacteriophages

• virulent or lytic phage

• temperate of lysogenic phage

conjugation

cell to cell contact via a pili

1st step in genetic transformation in bacteria

recipient cell takes up donor DNA

2nd step in genetic transformation in bacteria

Donor DNA aligns with complementary bases

3rd step in genetic transformation in bacteria

recombination occurs between donor DNA and recipient DNA

Virulent or lytic Phage

kills cell after infection

virus replicates in cell using host machinery

lyses the host cell to release progeny

Temperate of lysogenic phage

virus enters cell and integrates into host DNA

can exist in quiescent form while integrated in host genome

virus then replicates with the cell

it can excise out and leaves to infect another cell

Steps of lytic cycle

1. phage attaches to host cell and injects DNA

2. Phage DNA circularizes and enters lytic cycle or lysogenic cycle

3. new phage DNA and proteins are synthesized and assembled into virions

4. Cell lyses releasing phage virions

Steps of Lysogenic cycle

1. phage attaches to host cell and injects DNA

2. Phage DNA circularizes and enters lytic cycle or lysogenic cycle

3. Phage DNA integrates within the bacterial chromosome by recombination, becoming a prophage

4. Lysogenic bacterium reproduces normally

5. Occasionally the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle

Generalized transduction

occurs during the lytic cycle of viruses

random packaging of bacterial genes and proteins into virus

these generalized DNA can be carried to a new host

Specialized transduction

temperate phage: incorporates into hosts chromosome

must exist as a prophage

can spontaneously revert to lytic and excise out of the host DNA

• may include some of the hosts DNA into a new phage

these new specialized phage is carried to a new host

Steps of transduction of a bacteriophage

1. a phage infects the donor bacterial cell

2. phage DNA and proteins are made and the bacterial chromosome is broken into pieces

3. occasionally during phage assembly pieces of bacterial DNA are packaged in a phage capsid then the donor cell lyses and releases phage particles containing bacterial DNA

4. A phage carrying bacterial DNA infects a new host cell the recipient cell

5. Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells

Steps of Specialized transduction

1. prophage exists in galactose-using host

2. phage genome excises carrying with it the adjacent gal gene from the host

3. phage matures and cell lyses, releasing phage carrying gal gene

4. phage infects a cell that cannot utilize galactose (lacking gal gene)

5. Along with the prophage the bacterial gal gene becomes integrates into the new hosts DNA

6. lysogenic cell can now metabolize galactose

Conjugation

genetic exchange occurring through direct cell to cell contact and is mediated by a plasmid

requires the presence of a sex pilus

• encoded for by a fertility plasmid called the F factor

the f plasmid contains information to code for conjugal transfer and for autonomous replication

the donor cell carrying the F plasmid is male considered F+

the recipient cell is female and considered F-

Plasmids

small, extrachromosomal circular DS DNA

Independently replicates

Can exist in single or multiple copies

usually not essential for normal bacterial growth

conjugation allows for transfer between 2 cells

Plasmid types

1. conjugation function

2. resistance to antibiotics

3. resistance to heavy metals

4. resistance to bacteriophage infection

5. bacteriocin production

6. toxin production

7. virulence determinants

R factors

codes for resistance to antibiotics, very important medically

two groups RTF and r Determinant

Small segments of DNA which can move from one region of DNA to another region

transposable genes

RTF RESSISTANCE TRANSFER FACTOR

genes for plasmid transfer and replication

r Determinant

genes for detoxifying enzymes

usually carried on transposons

Conjugation continued

male or donor cell is F+

Female or recipient cell is F-

If cross an F+ cell with an F- cell it makes 2 F+ cells

• F+ transfers F factor (plasmid) to F-

• There is simultaneous replication and transfer of the plasmid

If F factor is incorporated into cells chromosome

• Hfr is High frequency recombinant

• this cell has the transfer information but it is integrated in host cells DNA

If we cross and HFr with and F- we get Hfr + F-

• because usually only a portion of F factor is transferred