Microbial Genetics:From Genotype to Phenotype

Nucleic acid synthesis

Protein Synthesis

Heredity

Genetic Recombination

Microevolution

Review Nitrogenous basedNTPBase pairingDNARNACoding strandTemplate strand

Notable differences in hereditary material in viruses, prokaryotes and eukaryotes

Important basis for modern methods of identification and disease diagnosis

Microbial Genetics

What does DNA do?Bacterial cell

DNA replication: Cell Division requires duplication of DNA

transcription

translationClones

RNA

DNA

protein

Large molecules of DNA are called chromosomes

Bacteria have circular chromosomes and sometimes additional small, circular pieces of DNA called plasmids

Replication of DNA

• Because DNA is double stranded replication can occur on each strand simultaneously in opposite directions

• Continuous, newly synthesized strand added in 5’ to 3’ is leading strand

• DNA synthesized from behind by the addition of Okazaki fragments is lagging strand, olignucleotides are still added to the 3’ end

DNA replication

Original double stranded DNA

Two new strands (green dotted lines) are created using a polymerase enzyme

The process of creating the new strands is called polymerization

DNA polymerization

3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…………….5’

5’ATCGAACGGAGACTTACTCTTA3’

DNA Polymerase

C A

A G

C

A

T

A

G

T

T

A

dNTPs are are added to the new strand (green) so that the dNTPs pair with the corresponding ones on the complimentary strand (black)

Sugar phosphate backbone Nitrogenous bases

DNA Polymerization

• Polymerization, requires DNA polymerase enzyme• Synthesis occurs only from 5’ to 3’ of new strand • dNTP incorporated into 3’ end of new strand by DNA

polymerase• Formula for polymerization:

(dNMP)nDNA +dNTP(dNMP)n+1DNA + PPi

• (dNMP)nDNA is the growing strand

• dNTP is the deoxynucleotide triphosphate

• (dNMP)n+1DNA is the growing strand after a dNTP is added

• iPP is inorganic diphosphate

Synthesis occurs on both strands of original DNA in opposite directions

Original strands are separated generating replication forks

5’

3’

Leading strandLeading strandLagging strands

Okazaki fragments

Nucleic acid polymerization

Nitrogenous base

Nitrogenous base

Nitrogenous base

Nitrogenous base

Nitrogenous base

Nitrogenous base

Nitrogenous base

Nucleotide triphosphate

5’

Polymerase enzyme

Inorganic phosphate

Transcription is synthesis of RNA from DNA

• NTPs (ATP, CTP, GTP, UTP) incorporated in to strand of mRNA (RNA polymerization)

• mRNA is complementary to the template strand of DNA• RNA polymerase enzyme is used

What is a gene?

DNA

rRNA

tRNA

mRNAprotein

mRNA synthesis occurs on the template strand (-) of original DNA

Original strands are separated where transcription is to begin

5’

3’

Synthesis of mRNA

Double stranded DNA has a coding strand (+) and template strand (-)

+

Synthesis of mRNA

3’TACCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…………….5’

5’AUGGAACGGAGACUUACUCUUA3’

RNA Polymerase

C A

A G

C

A

U

A

G

U

U

A

Template strand of DNA

AUG is start codon

Genetic Code for Building Proteins

Every three bases (triplet) of DNA corresponds to a codon of mRNA which corresponds to an anticodon in tRNA which bares a specific amino acid

Use the table in your book to provide the amino acids for the following DNA sequence on the coding strand :

CGTCCCGTC

Eukaryotes

E I E I E I E I E I E I E I E

E E E E E E E E

II I

II

I

I

DNA contains introns and exons

Exons have the code to build proteins

70S ribosome (prokaryotes)

30S subunit

50S subunit

21 different proteins

16SrRNA

23S rRNA and 5S rRNA

31 different proteins

Components of Prokaryote Ribosome

Ribosomes

The site of protein synthesis

Prokaryotes have 70S ribosomes

Eukaryotes have 80S ribosomes

Because ribosomes do such an important job drugs that inhibit them have drastic effects on the cell (many antibiotics and the toxin ricin inhibit protein synthesis by binding to ribosomes)

The genes for ribosomal RNA are often used to measure the similarity between organisms

16SrRNA gene for bacterial systematics

18SrRNA gene for eukaryote systematics

Translation

mRNA

Growing polypeptide connected by enzymatic linkage between amino acids

tRNA

ribosome

Example of regulation of protein synthesis: Enzyme Induction

Prom. Oper. Gene

mRNA

Pol.

repressor

Substrate binds to repressor allowing polymerase to transcribe mRNA

Enzyme synthesized

substrate

Enzyme acts on substrate

Example of regulation of protein synthesis: Enzyme Induction

Prom. Oper. Gene

Gene turned ‘off’Pol. repressor

In the absence of substrate, the repressor blocks polymerase enzyme

Note: the status of a gene being turned on or off is not heritable. While it allows individual organisms to adapt to changing circumstances, it does not effect the evolution of species as does mutation.

Mutation

• Point mutation -results from the replacement of a nitrogenous base effects may 1. be “silent” causing no change in the protein structure, or 2. result in altered protein with different performance or 3. produce protein that doesn’t work

• Frameshift mutation -results from the insertion or deletion of bases, has dramatic effects on protein, often deleterious.

the red fox ran out

the red fax ran out

thr edf oxr ano ut

After a point mutation the sentence might be:

After a frame shift mutation (deletion of letter e) the sentence might be:

The genetic code is analogous to written language

Suppose the sentence below is a genetic sequence:

Mutagens

• Chemicals that bind to DNA and affect the process of replication or transcription. Some naturally occurring chemicals are mutagenic, many industrial chemicals are mutagenic

• Radiation, including UV light, is mutagenic

Genetic Variation Results from Mutation

Most mutations are either harmful, or neutral, but sometimes they are beneficial.

If the mutations are not too harmful, they will be passed on to their progeny (offspring). This is the hereditary basis of evolution.

These heritable changes in a lineage or populations of organisms over generations contribute to micro-evolution

Antibiotic Resistance in Bacteria

• Bacteria can multiply rapidly into large populations that reach a stationary phase

• Antibiotic therapy or accidental ingestion of antibiotics acts as an agent for natural selection

• Single point mutation in DNA can lead to resistance in a single mutant bacterium

• Mutant ‘lost in crowd’ until antibiotic therapy kills off susceptible bacteria

• Mutant becomes dominant in population and gives rise to mutant clones

Variation by Mutation is Compounded by Genetic Recombination

• Sexual reproduction

• Bacterial transformation

• Bacterial conjugation

• Virus-mediated gene transfer

• Other transfer between symbionts

Sexual reproduction is genetic recombination

zygote

parent cells produced gametes by meiosis

gametes

Gametes fuse to form a zygote

2N diploid stage

1N haploid stage

egg sperm

gametogenesis fusion of gametes

Bacterial Transformation

Bacterium with gene for capsule

Bacterium without gene for capsule

Heat-killed bacterium disintegrate and release DNA into surroundings

Genes taken in and expressed

Conjugation

1. Plasmids may contain genes that allow a bridge (pilus) to form between two bacteria. Bacteria with these plasmids are F+

2. The plasmid from the F+ (donor) bacterium is replicated and transferred to the F- (recipient) bacterium

F+ F-

pilus

F+ F-

1. 2.

High frequency recombinations

F+

chromosome

Hfr+

Plasmid inserts into chromosome

Hfr+ recipient

recipient

Hfr+ DNA transferred into recipient and inserted into chromosome

Transposable elements (jumping genes)

• A mobile genetic sequence that can move from one plasmid sequence to another sequence or to a chromosome

• May result in the disruption of gene activity• Make up a large portion of Eukaryote DNA

Transduction

• Some viruses can incorporate their genetic material into the host’s chromosome

• When the virus is transferred from host to host, some of the host’s genes can be transferred along with the virus’s genes into the next host.

Bacterium with its chromosome containing viral (Prophage) DNA

Phage infects new bacterium bringing with it DNA from the previous one

Prophage DNA excised from host chromosome

Phage DNA inserts into new host chromosome along with bacterial genes

Transduction