Chapter 9

Chapter 9

Microbial Genetics

What is Genetics?

• The science of heredity

• Research in Genetics takes place on several levels

– Organismal

– Cell

– Chromosome

– Molecular

Where genetic material can be found?

• Genetic information is contained within genetic material – DNA and RNA

• It can be found in:

• Prokaryotic organisms

– Nucleoid (bacterial chromosom)

– Plasmids

• Eukaryotic organisms

– Chromosome

– Mitochondria

– Chloroplasts

– Plasmids

• Viruses

– DNA

– RNA

Some definitions

• Chromosome – Cellular structure -packaged DNA molecule

• Gene – A segment of DNA that codes for functional product (protein)

• Genotype – The sum of all genes -Genetic make-up

• Phenotype - Manifestation of genotype (ability to perform particular chemical reaction)

The size and packaging of genomes

• E.coli 4,288 genes

• Human cell: 20,000 – 25,000 genes

• Size of the cell of E. coli – 1 mm (diameter)

• Size of the stretched out DNA – 1mm (1000 times longer)

DNA molecule is tightly packed in the form of chromosome

Structure of DNA

• DNA molecule – double helix

• Composed of nucleotides

• Nucleotides composed of

– Bases

• Purine (adenine, guanine)

• Pyrimidine (cytosine, thymine)

– Deoxyribose sugar

– Phosphate group

• Single strands of DNA have opposite orientation (3’ and 5’)

• G-C held together with 3 hydrogen bonds

• A-T held by 2 hydrogen bonds

DNA Replication – overall process

· Topoisomerase unwinds the double helix of DNA

· Helicases break hydrogen bonds

· Bases of single stranded DNA exposed

· Replication fork is formed

· Synthesis of the new strand by attachment of complementary nucleotides

Other enzymes involved in DNA replication

• Helicase – Untwists the DNA helix by braking the H bonds

• Primase - Synthesis of RNA primer

• DNA polymerase III – adding bases to the new DNA

• Ligase - joining of the of DNA fragments

• Topoisomerase I, II - Supercoiling

Details of replication

• Helicase opens the double helix

• Complementary nucleotides attach themselves to the exposed bases

A-T C-G

• DNA polymerase III joins the added nucleotide into a growing DNA strand

• Semiconservative replication (double stranded DNA contains one old and one new strand)

DNA Replication

• Leading strand is synthesized continuously

• Lagging strand is synthesized in pieces (Okazaki fragments)

• The strands are synthesized in the 5’ to 3’ direction

• DNA polymerase I removes RNA primers

• Lygase joins the fragments

The Flow of Genetic Information

• DNA Replication – reproduction of cells

• Transcription – information contained within DNA is copied è RNA

• Translation – polypeptides synthesized from RNA nucleotide sequences è amino acid sequence of protein

The difference between DNA and RNA

DNA RNA

Sugar component: desoxyribose ribose

G -----------C

C------------G

T-------------A

A------------ U (uracyl)

Transcription

• Synthesis of RNA from DNA as a template

• Messenger RNA (mRNA) is formed by transcription of a portion of DNA (gene)

• Sequence of nucleotides from DNA are rewritten in RNA

Process of transcription

• RNA polymerase binds to the promoter

• Unwinds the double helix of DNA

• One DNA strand acts as a template for synthesis of RNA

• RNA polymerase puts free nucloetides together forming RNA chain

• As the new RNA grows, polymerase moves along the DNA

• When the RNA polymerase reaches the sites called terminator - the transcription ends - RNA polymerase and new RNA strand are released

Translation

• Is the process of translating nucleic acid language into the language of proteins (sequence of amino acids)

• Codons are groups of three nucleotides (mRNA)

• The sequence in the codon determines which amino acid will be incorporated into a protein

• Translation is taking place in ribosomes

The beginning of protein synthesis

• Ribosom moves along mRNA and reads the codons

• Transfer RNA (tRNA) has a sequence of three bases – anticodons complementary to codons

• For each amino acid there is a specific tRNA

• The first tRNA recognizes the start codon and brings methionine

Elongation and termination

• Peptide bonds are formed between amino acids -polypeptide is formed

• Blank tRNA discharged

• When nonsense codon is reached, translation is terminated

• Ribosome is separated into its subunits

Simultaneous transcription and translation

• This is a fast process

• More than one ribosome reads one mRNA molecule

• Most genes (75%) are constitutive – their products are produced constantly

• Other genes are regulated so that they are transcribed and translated only when needed

Transcription and translation in Eukaryotes

• The introns – non-functional segments of DNA) are transcribed but not translated

• The genes need to be processed

• The introns are cut out

Mechanisms of Genetic Control

• Repression

– Inhibition of gene expression

• Mediated by a protein – repressor

• Induction

– Induction of gene expression (transcription)

• Inducer – a substance that induces transcription

Mechanisms of Genetic Control

• Repression

– Inhibition of gene expression

• Mediated by a protein – repressor

• Induction

– Induction of gene expression (transcription)

• Inducer – a substance that induces transcription

Operon model of gene expression

Inducible operon

Lactose operon (regulates lactose metabolism) - must be activated by inducers

Functional gene consists of:

Regulatory gene

Operon

Control region

Promoter - RNA polymerase initiates transcription

Operator – go or stop signal

Structural genes

- b-galactosidase

- lac permease

- transacetylase

Changes in the Genetic Material

• Mutation – change of the genetic code

• Horizontal gene transfer

– Transformation

– Conjugation

– Transduction

Mutation

• During the replication of DNA - an error in base sequence may occur - mutation

• It can be:

– Spontaneous

– Induced

• Mutation can have beneficial or deleterious (lethal) effect

– beneficial - mutant enzyme - enhanced activity (rare)

– lethal - lost activity of the enzyme (almost always)

Categories of mutation

• Point mutation – substitution, insertions, and deletions of a single base in the DNA

• Missense mutation – substitution of one amino acid (Inactive or reduced activity protein)

• Nonsense mutation - creating a stop codon in the middle of protein

• Frameshift mutation -deletion or insertion of a DNA fragment; causes changes in many amino acids

Mutagens

• Chemicals - nitrous acid (converts adenine into form that pairs with C instead of T)

• Nucleoside analogs (molecules that are structurally similar to normal bases)

• Radiation-

– X-rays, gamma rays (errors in replication; physical damage of DNA)

Transformation

• Fragments of the DNA are transferred from one microorganism to another (competent) – incorporated into genom

• Experimentally shown (but not understood) by Frederick Griffith in England 1928

Conjugation

• “Sexual contact” between two mating cells

• Donor cell has sex pilli and F+ (fertility factor) a gene located on the plasmid

• DNA is transferred from one cell to another through sex pillus

Plasmids

• Small circular self-replicating DNA elements

– Conjugative plasmids

– Enable survival under challenging conditions (exotic substrates – hydrocarbons, etc)

– Production of toxins (kill other bacteria)

– Resistance factors (antibiotics)

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