MIC 102 A01 - Final

Mutations

Change in genetic material

• Change in DNA → change in RNA → change in protein

• May be neutral, beneficial, or disadvantageous

• Beneficial mutations give rise to diversity, adaptation to environment

• Natural selection removes individuals with disadvantageous genotypes

• IMPORTANT: Mutations arise BEFORE selection!

Disadvantageous errors will be most costly to a cell and its progeny if they occur during….

A. DNA replication

B. Transcription

C. Translation

A. DNA replication

Redundancy, wobble allows for silent mutations (encode the same amino acid)

E.g., 6 different codons for Leucine, a highly abundant amino acid:

• CUU

• CUC

• CUA

• CUG

• UUA

• UUG

Missense mutations can have a range of impacts on protein structure and activity

Impact of amino acid substitution depends on:

• Similarity of WT and mutant amino acids

• Role of WT amino acid in catalytic activity, structure, interactions with other proteins

The Frequency of Mutation

Spontaneous point mutation

• DNA polymerase (DNAP) incorporates the wrong nucleotide every ~1 in 10^6 bp

• Bases can spontaneously deaminate, causing them to be misread during DNA replication

Dinal mutation rate is ~1 in 10^10 bp (after proofreading, mismatch repair, etc)

• Mutagens increase the mutation rate 100-1000x

Types of mutagens

• Radiation: Ultraviolet (UV), ionizing (x-rays, gamma)

  • UV causes pyrimidine dimers; e.g., neighboring thymine or cytosine residues on the same strand will be covalently linked

• Chemicals: e.g., base analogs

  • 5-Bromouracil (5-BrU) is an analog of T that can pair with A or G

  • Result: T:A → C:G transition

  • Biological “mutator” strains that lack proofreading or repair

  • Recombination events

  • Insertion of transposons, viral genomes

Recombination

Mixing of genetic material

• Occurs between regions with identical sequence

• The scale is variable; entire genes or operons can be exchanged

• Can result in a new strain that is different from the donor and recipient

Transposons

Cut-and-paste transposition model

• Transposon elements:

  • Inverted repeats (IR)

  • Transposase

  • Other genes (e.g., AbxR)

How are genes passed between bacteria?

Conjugation: Single strand DNA gets passed from one living bacterium to another through a pore/channel

Transformation: “funeral pass”

• Bacterium takes up naked DNA (from lysed cell?)

• Recipient maintains circularized replicon or incorporates the DNA into its genome

Transduction: “viral pass”

• A bacteriophage packages bacterial DNA from infected (lysed) cell and transfers it into new host bacterium during infection

Conjugation

A donor cell passes DNA to a recipient

• F Factor: “fertility” plasmid with origin of transfer & pilus assembly genes

Transformation

“Naked” DNA is taken up, which “transforms” the phenotype

• Frederick Griffith (1879-1941): Bacteriologist studying pneumonia

• Streptococcus pneumoniae: Still responsible for ~22,000 deaths/year, particularly among the

• Relative virulence of S. pneumoniae strains:

  • “Rough” - less virulent

  • “Smooth” - more virulent

Barriers to transformation:

• DNA must cross permeability boundaries (CM, OM, PG)

• Negatively-charged DNA must get close to the negatively-charged bacterial cell membrane

Competence: the ability to actively take up free, foreign DNA; often transient and inducible

Natural competence:

• ~1% of bacteria readily import DNA from their environment (in vitro) under permissive conditions

• Inducible by high [cell], damage

Artificially-induced competence:

• Used in a laboratory to transform bacterial cells

• Makes transient pores in the cell membrane

• Can transform linear or circular DNA

• Methods to induce competence:

  • Electroporation

  • Chemical competence:

    • A high concentration of cations (usually Ca²+)

    • Shock the cells with heat

Conclusions from Griffith’s experiment

The “transforming principle” (DNA) released from dead cells was incorporated by living cells, resulting in their genetic transformation.

Therefore:

• Traits can be acquired

• DNA can be exchanged between organisms

• Bacteria are a powerful research model to study basic principles

What is genetic engineering?

• Refers to the direct manipulation of DNA to alter an organism’s characteristics in a particular way

• This can range from changing one single DNA base to deleting or inserting a whole region of DNA

• For example, this can be used to produce more efficient metabolite production or drug biosynthesis

Steps to genetically engineer a bacterium to synthesize a foreign gene product (e.g., human insulin)

1. Purify [plasmid]

2. [Make many copies] of target gene DNA (insert)

3. Use [restriction enzymes] to “cut” insert & plasmid

4. Ligate cut insert into cut plasmid with [DNA Ligase]

5. Transform cells with recombinant plasmid

   • Perform artificial transformation with recombinant plasmid. Then plate treated cells onto media with selection (e.g., antibiotic) to select for transformants carrying the selectable marker (e.g., abx^R)

6. Induce (activate) expression of gene

7. Purify product

8. Make $$$ and save lives

• [Re-purposed from nature]

Example of a cloning vector: A plasmid

Vectors:

• Small pieces of DNA that can be stably maintained by the recipient

• Foreign DNA fragments can be introduced that encode new properties to the recipient

• Called “vectors” because they have direction; transmit genes from one organism to another

Plasmid properties:

• Origin of replication

• Selectable marker (Abx^R)

• Restriction enzyme cut sites

• High copy number

• Small size

Setting up a PCR “reaction”:

Reagents:

• Water

• Buffer - to keep the pH near neutral, provide KCl (K+)

• dNTPs = deoxyribonucleotides (ATP, GTP, CTP, and TTP)

• Heat-stable DNA polymerase; e.g., Taq DNAP, isolated from the thermophilic bacterium Thermus aquaticus, which has an optimal growth temp ~70C and a maximal growth temp ~80C

• MgCl2 - Mg²+ is a required co-factor for DNAP

• Primer (chemically-synthesized deoxyribo-oligonucleotides/”oligos”)

• Template (DNA)

Materials:

• Microfuge (test) tubes

• Thermocycler machine

Polymerase Chain Reaction (“Amplification”)

~30 cycles

• Denaturation of dsDNA 95C

• Annealing of primers (~60C sequence specific)

• Extension of primers

Restriction enzyme (RE) “digest” of insert & vector

REs are endonucleases that “cut” (hydrolyze the phosphodiester bond of) the phosphate-sugar backbone of dsDNA at specific sequences

• Ex: EcoRI, recognition sequence, GAATTC; cut site: G|AATC

Restriction-Modification (R-M) system protect against viruses

• The activity of restriction enzymes (REs) in the bacterial cytoplasm “restricts” the replication of bacteriophage to certain hosts

• To protect against phage infection: a RE cuts double-strand DNA (e.g., viral genomes) that carry the RE’s recognition sequence, which prevents replication of the viral genome

• To protect host DNA with the recognition sequence: A methylase that recognizes the same DNA sequence as its RE partner modifies the DNA by adding a methyl group; methylation at its recognition site inhibits the partner RE from cutting the DNA.

• RE + methylase = restriction-modification (R-M) system

R-M system

Bacterial immune system

• REase = restriction endonuclease (enzyme)

• MTase = methyltransferase (methylase)

CRISPR-Cas: Immune system in bacteria

Clustered regularly interspaced short palindromic (a word, phrase, or sequence that reads the same backwards as forward, e.g., madam or nurses run.) repeats

1. CRISPR array is assembled on bacterial chromosome by the insertion of phage seqs (spacers) and a CRISPR-specific repeat seq

2. CRISPR array is transcribed to produce precursor RNA (pre-crRNA)

3. crRNA that match viral sequence recruits Cas9 (CRISPR-associated nuclease) which cuts the viral genome, preventing phage replication

   • Task: Remember this phage to fight off this phage

Regulation of enzyme amount

• Microbes are frequently substrate limited

• Transcription and translation are coupled

• Microbial mRNA are short lived

• Genes are organized in operons

Regulation of enzyme activity

Non-covalent modifications:

• Competitive inhibition

• Non-competitive = allosteric

Covalent modifications:

• Phosphorylation

• Methylation

• Acetylation

• Others

Inhibition of folate biosynthesis

Dihydropteroate diphosphate + para-aminobenzoate (PABA)

→ Dihydropteroate reductase (No sulfonamides)→ Dihydropteroate

→ Dihydrofolate → Dihydrofolate reductase → Tetrahydrofolate*

• Tetrahydrofolate is a co-enzyme required for nucleic acid, amino acid synthesis and other essential cell functions

Regulation of flux in central metabolism

Cells regulate flux (flow through the pathways) in response to:

• Concentration of reductant, precursors, & building blocks

• Energy status

Enzyme activity regulated by allosteric activation/inhibition

• High concentration of substrate?

  • More flux through pathway, more enzyme activity

• High concentration of product?

  • Less flux through pathway, less enzyme activity

The cell maintains a high [ATP] relative to [ADP] and [Pi]

• ATP ←→ ADP + Pi

• ATP ←→ AMP + PPi

  • If [ATP] is too low, more flux through fueling is needed

CRP-cAMP activator (Glucose -)

• If glucose is NOT being transported into the cell, cyclic AMP (cAMP) is produced

• cAMP binds to the cAMP Receptor Protein (CRP; aka Catabolite Activator Protein, CAP), which increases its affinity for its specific binding site

• cAMP-CRP binds and enhances RNAP binding to the promoter

CRP-cAMP activator (Glucose +)

• Glucose is transported into the cell → no cAMP produced → CRP does not bind promoter or activate transcription

• There is global regulation of many operons by CRP (~200 transcriptional units in E. coli) including lac and other catabolite-utilization operons (called “catabolite repression” or the “glucose effect”)

Expression dilemma

Consider this paradox:

• Lactose must be transported into the cell in order for its transporter to be synthesized

• Lactose must be converted into allolactose by beta-galactosidase in order for beta-galactosidase to be synthesized