Molecular Biology: Transcription and Translation

Microbiology Study Notes

• Microbiology is the scientific study of microorganisms, being unicellular, multicellular, or acellular. It includes virology, bacteriology, protistology, mycology, immunology, and parasitology.
• Microbes are everywhere, originating all forms of life, and comprising the majority of biomass on Earth. They exist virtually everywhere where water is available, often under the Earth's surface.
• Microbes are essential to the food chain and affect the climate. They also are symbiotic with animals, humans, and other microbes; simply essential for life.
• Pathogenic microorganisms significantly affected human populations historically, leading to death in 1900. While they remain important, infectious disease is less significant now.
• Infectious diseases, accidents, perinatal and maternal diseases and others are significant causes of death in both developed and developing countries. Infectious disease is a leading cause of death in developing countries.

Milestones of Microbiology

• Antoni van Leeuwenhoek (1632-1723): A natural philosopher, he made microscopes and observed bacteria.
• Louis Pasteur (1822-1895): Demonstrated infectious diseases are caused by microbes (Swan-necked Flask Experiment), developed pasteurization, and vaccines for diseases like anthrax and rabies.
• Robert Koch (1843-1910): Developed postulates for proving cause and effect in infectious diseases. Discovered that bacteria can cause diseases like anthrax, tuberculosis, and cholera, and received a Nobel Prize.

The Diversity of Microorganisms

• Three domains of organisms exist: Bacteria, Archaea, and Eukarya.
• Organisms are grouped by phylogenic tree.
• Hyperthermophiles are prokaryotes that reach their optimal growth at a temperature of 80°C or higher.
• Ribosomal RNA (rRNA) gene sequencing is used to create a phylogenetic tree and understand relationships between organisms.

Size of Microorganisms

• Microorganisms range in size from 0.2 μm to 50 μm.
• The ratio of surface area to volume in cells decreases as the size of the cell increases and this directly affects the cell's metabolism.

Properties of all cells

• All cells have a cytoplasmic membrane, cytoplasm, a genome made of DNA, and ribosomes.
• All cells use encoded information in DNA to make RNA and protein.
• All cells take up nutrients, transform them, conserve energy and expel wastes.
• 2 major categories of metabolism: catabolism and anabolism.
• All cells undergo growth using information to convert nutrients into new cells.
• Cells evolve through mutations in DNA.

Properties of Some Cells

• Some cells can form new structures such as spores.
• Cells interact with each other via chemical messengers.
• Some cells are capable of self-propulsion.
• Cells exchange genes by various mechanisms.

Gram Staining

• Gram-positive bacteria have a thick peptidoglycan layer and no outer membrane.
• Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane.

Cell Wall Structure

• The backbone of peptidoglycan is formed from N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).
• Peptidoglycan provides protection and shape to bacteria.
• Penicillin-binding proteins (PBPs) catalyze the polymerization of NAG and NAM and crosslinking of peptide bridges, interrupting peptidoglycan synthesis.
• Lysozyme also breaks down these connections.
• Archaea have pseudopeptidoglycan and S-layers, similar in structure to peptidoglycan, but using N-acetyltalosaminuronic acid instead of muramic acid.

Outer Membrane of Gram-Negative Bacteria

• Gram-negative bacteria have an outer membrane that contains lipopolysaccharides.
• Ca2+ ions are required to maintain the stability of the lipopolysaccharide layer.

Cytoplasmic Membrane

• The cytoplasmic membrane is a selectively permeable membrane, important for maintaining the cell's environment.
• This membrane is a fluid mosaic model consisting mostly of phospholipids.
• The composition of the membrane is homeovcous, adapting to environmental conditions to preserve fluidity.
• There are 3 major mechanisms for moving molecules: passive diffusion for small molecules and water; facilitated diffusion, and active transport.

Surface Structures: Flagella, Fimbriae and Capsules

• Flagella enable movement, distributed over the whole surface (peritrichous), or are located at a single pole (monotrichous), or at both (amphitrichous).
• Fimbriae are used to attach to the surfaces of host cells, aiding in colonization and potential infection.
• Capsules are polysaccharide structures that protect bacteria from phagocytosis.

Endospores

• Some gram-positive bacteria can form endospores, which enables them to survive harsh environments and conditions.
• This protects the bacterium from environmental influences.

The Hallmarks of Cellular Life

• For cellular self-replication sufficient energy and genetic material must be provided or replicated beforehand.
• Gene expression (transcription and translation) produces proteins for cellular function.

Information Storage in DNA and RNA

• DNA is a double helix (anti-parallel), composed of deoxyribose, phosphate residue, and bases.
• RNA is a single strand, composed of ribose, and phosphate residue, and bases.
• DNA & RNA bases differ (uracil instead of thymine).
• The sequence of nucleotides in mRNA translates to proteins.

DNA Replication

• DNA is replicated in the 5' to 3' direction.
• Multiple enzymes are involved in the process: DNA polymerase, DNA ligase.

Transcription (DNA → RNA)

• RNA synthesis occurs at specific sites (promoters) on DNA.
• The sigma factor is required for the polymerase to find promoters.
• The RNA polymerase moves down the DNA strand, separating the double helix temporarily.
• Transcription proceeds until a termination site is reached, releasing the mRNA and the polymerase.

Translation (RNA → Protein)

• Ribosomes translate RNA into proteins.
• Three nucleotides code for one amino acid, read by the tRNA.
• The sequence of nucleotides is read in consecutive groups of three.

Amino Acids: Building Blocks of Proteins

• 20 amino acids are common to proteins.
• Amino acids have an amino group, a carboxyl group and a characteristic side group ("R").
• These groups are divided into 4 categories: ionizable: acidic, ionizable: basic, nonionizable polar, and nonpolar.

Bacterial Growth: Binary Fission

• Bacteria reproduce by binary fission.
• This involves DNA replication, cell elongation, septum formation, and the separation of the two daughter cells.
• Bacterial growth is characterized by distinct phases: lag phase, log phase, stationary phase, and death phase.

Abiotic Parameters Affecting Bacterial Growth

• Temperature affects enzymatic reactions.
• Osmolarity affects water balance.
• pH affects enzymatic activity.
• Oxygen affects some cultures.
• Water activity affects microbial growth.

Microbial Growth in Different Environments

• Bacteria can grow at varied temperatures; thermophiles grow at higher temperatures, mesophiles at moderate temperatures, and psychrophiles at lower temperatures.
• Factors that influence survival are enzyme stability, membrane fluidity, and the structure of the protein.

pH and Growth

• Most bacteria can grow over a wide pH range, but pHi (intracellular pH) tends to remain near neutral.
• They do this using enzyme-catalyzed reactions (consumption of protons/acids).
• Cells alter lipid composition of the cytoplasmic membrane to reduce permeability to protons during extreme pH conditions.

Osmolarity

• Bacteria alter their internal solute concentration or pump substances into cells to maintain a consistent internal environment when exposed to high or low external solute concentrations.
• Compatible solutes are water-soluble sugars, alcohols, and amino acid derivatives that maintain osmotic balance.

Water activity

• Bacteria require relatively high water activity to thrive.
• Water activity is measured as Aw, which may vary from 0 to 1.
• Aw values below 0.95 are unfavourable for many bacteria.

Methods of Preventing Microbial Growth

• Heat sterilization (autoclave)
• Heat (pasteurization)
• Ionizing radiation
• Filtration
• Chemical agents (disinfectants)

Chemical Growth Control: Antibiotics

• Antibiotics interfere with bacterial growth through various pathways depending on their type.
• Antibiotics can be bacteriostatic, bacteriocidal, or bacteriolytic, by affecting protein synthesis, cell wall synthesis, folate synthesis, nucleic acid synthesis and cell membrane.

Resistance to Antibiotics

• Many pathogens are becoming resistant; preventing and treating infections is becoming an increasing challenge.
• Modifications to the bacterial cells/structures, like the production of efflux pumps, often lead to resistance.

Mechanisms of Antimicrobial Drug Resistance

• Efflux pumps, inactivation, target modification, and target bypass are some resistance mechanisms bacteria can use.

Nutritional Requirements for Microbial Growth

• Required nutrients include carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, potassium, magnesium, sodium, calcium, iron, as well as trace-elements and vitamins.

Culture Media for Bacteria

• Defined media is specifically composed media for a specific strain, composed of specific components and chemical quantities.
• Complex media contain various macro- and micronutrients that enable various organisms to grow.

Cellular Respiration: The Citric Acid Cycle and Electron Transport Chain

• Respiration pathways, such as aerobic respiration, produce ATP.
• The citric acid cycle breaks down acetyl-CoA, producing energy.
• The energy from the electron transport chain is used to generate a proton gradient that drives ATP synthesis.

Types of Bacterial Metabolism

• Chemoorganotrophs: use organic compounds for energy.
• Chemolithotrophs: use inorganic compounds for energy.
• Phototrophs: use light for energy.
• Heterotrophs: use organic compounds as carbon sources.
• Autotrophs: use carbon dioxide to produce organic compounds.

Chemolithotrophic Metabolism

• Chemolithotrophs use oxidation of inorganic compounds to produce energy and synthesize cellular components.
• Several types of chemolithotrophic metabolism exist and use reactions where e.g., ammonia or nitrite are oxidized to produce energy for the cell.

Phototrophic Metabolism

• Phototrophs use light as a source of energy to produce ATP.
• Photosynthesis can produce oxygen as a byproduct (oxygenic photosynthesis) or not (anoxygenic photosynthesis).
• Several important types of phototrophs include cyanobacteria, purple sulfur bacteria, and purple nonsulfur bacteria.

Regulation of Gene Expression

• The product of a gene may be an enzyme or other protein directly involved in cell function.
• Synthesis of gene product can be initiated, regulated or inhibited through transcriptional and/or translational control.

Mechanisms of Gene Regulation: Operons

• Operons are clusters of genes that are transcribed together; coordinated regulation of expression.
• Repressors prevent transcription, while activators promote it.

Regulation of Gene Expression Mechanisms

• small RNA molecules and riboswitches regulate translation.
• Co-transcriptional and co-translational processing of RNA influences function.

Post-Translational Regulation: Feedback Inhibition

• Synthesis of polypeptide chains and subsequent modifications may be controlled and regulated at various points.
• Enzymes can be inhibited, especially the initial enzyme in a sequential conversion pathway, by end product through allosteric feedback inhibition.
• Allosteric feedback inhibition prevents the production of excess product.

Post-Translational Regulation- Covalent Modification

• Covalent modification, such as the addition or removal of chemical groups to proteins (e.g., adenylation).
• This can change their shape and functionality
• Regulation of enzyme activity often involves covalent modification

Viral Replication

• The basic viral replication cycle involves adsorption, penetration, uncoating, synthesis, maturation, and release (sometimes called assembly).

The Diversity of Viral Genomes

• Viral genomes can be DNA or RNA, and can be single-stranded (ss) or double-stranded (ds).
• Viral genomes are categorized into different groups.

Bacterial Phage Receptors

• Many bacteriophages have receptors on their surface where they may adhere to bacterial cells to initiate infection.
• These receptors may be proteins, polysaccharides, etc..

Bacteriophages Replication Cycle

• Several methods, such as lytic and lysogenic cycles exist for viral replication.
• The lytic cycle results in the lysis and destruction of the host cell while providing new infectious phage virions compared to the lysogenic cycle, where the phage DNA integrates into the host genome, without immediately destroying it.
• Some key viral components are present in phage replication, such as genomes, endolysins.

Timeline of Events During Lytic Phage Infection

• Early viral proteins are produced to initiate gene expression and genomic replication (e.g., nucleases, DNA polymerase, and other phage-specific sigma factors).
• Late viral proteins are produced for virion assembly and release (e.g., phage structural proteins and lysozyme).

Phage Lambda: A Temperate Phage

• A Temperate Phage establishes lysogeny by integrating its DNA into the host genome; producing prophages which can be excised at a later time to establish a lytic cycle.
• Regulatory proteins (Cro, ci) determine whether a lytic or lysogenic cycle takes place
• The process involves integration events, excision (cleavage of prophage DNA from the host chromosome), DNA replication.

Phi-X174: Phage with a Single-Stranded DNA Genome

• Phi-X174's small genome features a single-stranded DNA genome that is partially duplicated via a self-replication mechanism and provides an advantage in DNA sequencing.

Phage M13: A Filamentous Phage

• Phage M13 is a filamentous phage which does not kill the host cell; it replicates and leaves host cells via exocytosis.
• Bacteria may use M13, and other similar phages, for information exchange.

Phage MS2: A Small (+) RNA Phage

• The phage MS2's genome is a single-stranded (+) RNA capable of being translated into proteins without a DNA intermediate.

The Diversity of Animal Viruses

• Viruses are characterized by their highly diverse structures and relative sizes, and vary by genome type.

The Lytic Human Herpesvirus Life Cycle

• Binding, entry (fusion or endocytosis), nuclear transport, nuclear entry, gene expression, DNA replication, packaging, and egress are the key steps.

Poliovirus

• The Poliovirus has a single-stranded (+)RNA genome, and is part of the Picornaviridae family.
• The Poliovirus has a protective capsid that protects against environmental stresses.

Structure and Function of a Retrovirus

• Retroviruses, such as HIV, have an RNA genome.
• This RNA genome is transcribed into DNA (with reverse transcriptase).

The Lifecycle of Human Retroviruses

• Entry via specific receptors (for cells like CD4 /CCR5/CXCR4) on the plasma membrane.
• Then the viral genome is transported through nuclear pores.
• Reverse transcription produces viral DNA, which is integrated into the host genome to produce proviral DNA.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

• Coronaviruses belong to the family of enveloped (+)RNA Viruses.
• Their genomes are extremely large.
• The virus infects cells by binding to receptors like ACE2.
• The virus can use an endocytosis pathway.

Viroids

• Viroids are small, single-stranded circular RNA molecules
• Viroids do not code for proteins and depend on the host enzymes for replication and regulation of expression, to cause plant diseases.

Prions

• Prions are misfolded and aggregated proteins known as prions, which are uniquely infectious and very resistant to usual measures.
• Prions cause rapidly progressive, incurable neurodegenerative disorders.
• They produce abnormally folded prion protein (PrPSc) which catalyzes the refolding of normal prion protein (PrPc) into the PrPSc form.

The Microbiome

• The microbiome is the collection of all microorganisms (bacteria, fungi, virus) that live in or on a multicellular organism.
• Microbial populations play a role in human health (including synthesis of vitamins, digestion, amino acid production) and health disorders.

Biochemical/Metabolic Contributions of Intestinal Microorganisms

• Intestinal bacteria synthesize many vitamins and amino acids, fermenting sugars lead to gas production, and produce various short-chain organic acids.

Fermentation in the Colon of Lean and Obese Mice and Transfer of an Obese Condition by Fecal Transplant

• Obese mice contain more firmicutes (bacteria), compared to lean mice, that produce more H2 during fermentation.
• Volatile fatty acids (VFAs) are produced during fermentation.

Physical, Chemical, and Anatomical Barriers Against Infection

• Barriers exist and protect against pathogen invasion in the body (chemical, mechanical, epithelial barriers and the microbiome).
• These barriers stop entry of pathogens.

The First Step of Infection: Bacterial Adherence

• Adherence is the initial step of bacterial infection.
• Bacterial structures (like capsules, fimbriae, flagella and pili) assist in adhering to the host cell.
• Biofilms may aid in establishment of bacteria.

Enzyme Virulence Factors of Some Gram-Positive Bacterial Pathogens

• The activity of some enzymes, as virulence factors (example hyaluronidase, coagulase and streptokinase), may destroy tissues (affect host cells), and break down tissues (affect hosts), leading to bacterial spread in the host.

Pathogenicity Islands and Plasmids in Salmonella

• Pathogens like Salmonella have various virulence factors in various islands or plasmids.
• These structures contain genes encoding factors for pathogenicity.

Some Exotoxins and Cytotoxins

• Exotoxins and cytotoxins are protein toxins secreted by bacteria.
• Superantigens trigger a massive immune response, unlike other exotoxins, affecting whole populations of T-cells.

AB-Toxins

• AB toxins consist of two parts: A unit (toxic) and B unit (binding).
• Diphtheria toxin exemplifies this class, where toxin A catalyzes ADP ribosylation and inactivates elongation factor affecting eukaryotic translation, resulting in cell death.

Clostridium Botulinum Toxin

• C. botulinum produces a potent neurotoxin,
• This toxin blocks acetylcholine release at neuromuscular junctions prevents muscle contraction.

The Cholera Toxin Excreted by Vibrio Cholerae

• Vibrio cholerae is an anaerobic comma-shaped bacterium commonly found in brackish or saltwater
• It produces cholera toxin (CT) that results in severe diarrhea.

Cytotoxins (Cytolytic Toxins)- The Staphylococcal a-Toxin

• The a-toxin is often secreted by Staphylococcus aureus.
• Staphylococcal a-toxin is a pore-forming cytotoxin creating transmembrane pores into host cells and causing cell lysis.

Exotoxins vs. Endotoxins

• Exotoxins are secreted proteins, and are highly toxic in very small quantities, often causing specific reactions.
• Endotoxins are LPS components that are part of the outer membrane of gram-negative bacteria, causing fever and general reactions even in large amounts.

Basics of Immunology

• The immune system has two-pronged defenses: innate and adaptive.
• Innate immunity acts rapidly and generally against a broad spectrum of pathogens; it doesn't have immunological memory.
• Adaptive immunity involves more specific targeting and has immunological memory, enabling quicker future responses to encountered pathogens.

The Blood and Lymphatic Systems

• The blood and lymphatic systems serve as the transport and circulatory systems for cells and proteins within the body.
• Major blood components include erythrocytes (red blood cells), leukocytes (white blood cells), lymphocytes, and granulocytes/monocytes.

Microbial Invasion and the Innate Immune Response

• Infection leads to microbial invasion from tissues.
• Pathogens are recognized via the pathogen-associated molecular patterns (PAMPs), allowing the immune response pathway to be triggered.
• Phagocytes (macrophages, neutrophils) are attracted and activated using chemokines and phagocytose invading pathogens.
• Cytokines are released to direct and regulate the innate immune response.

Signal Transduction in Innate Immunity

• Signal transduction pathways are frequently generated by PAMP binding to pattern recognition receptors (PRRs) on the surface of phagocytes.
• This sets off a series of chemical modifications to proteins which will ultimately lead to a response, like a quicker or more controlled immune response by phagocytes.

Phagocytosis

• Phagocytosis is the biological process of engulfing and eliminating pathogens (like bacteria) by phagocytes (immune cells)..
• Toxic oxygen compounds are created (e.g., hydrogen peroxide, hydroxyl radical, hypochlorous acid, superoxide anion, singlet oxygen, nitric oxide).
• Toxic compounds are used to kill pathogens.

Inflammation and Fever

• The release of inflammatory cytokines (IL-1, IL-6, TNF-α) induces fever.
• These chemicals cause the brain's temperature control center to produce prostaglandins, which increase body temperature.

Adaptive Immunity

• Adaptive immunity includes T cells and B cells.
• T cells are a type of immune cell involved in cell-mediated immunity, while B cells produce antibodies.

Antigen Presentation

• MHC (major histocompatibility) complexes are integral proteins utilized in adaptive immune responses to present antigens to their corresponding receptors in order to activate an adaptive response.
• MHCs exist in 2 forms (I and II) that present different sources, cellular proteins or exogenous proteins, and present them to different types of T cell receptors.

T-Helper Cell Subsets

• Th cells (T helper) aid in antibody immunity and cell-mediated immunity by producing various cytokines.
• There are subsets of Th cells, such as Th1, Th2, and Th17, each with specific functions.

T-Helper Cell Subsets and T-cell Selection and Clonal Deletion

• Immune cells (T cells) are selected and matured, allowing them to respond to infection and not to their own body tissues (self-tolerance).
• This process is necessary to prevent autoimmune responses, where immune cells attack the host's own tissues.

B Cell Clonal Selection and Expansion

• B cells, which recognize and respond to specific antigens by producing antibodies, are selected and expanded to generate clones with the same antigen-specific antibody, via clonal selection.
• Antibodies binding to antigens leads to continued proliferation of clones of the responsive B cells.

Properties of Human Immunoglobulins

• Antibodies (Immunoglobulins) are Y-shaped proteins.
• They have variable regions that bind to specific antigens and constant regions that mediate interactions with other parts of the immune system.
• There are different classes, with differing activities in the body.

Active and Passive Immunity

• Active immunity is the immunity that an organism develops during an infection and results in immunological memory responses that can be recalled for future exposure.
• Passive immunity is the form of immunity received or transferred via antibodies.

Immunoglobulin Gene Rearrangement in Human B Cells

• Immunoglobulins have genes that rearrange in various ways to generate a large diversity for antigen-recognition receptors and to give each B cell unique specificity.
• This diversity is necessary to generate a variety of immune responses to combat various pathogens and to prevent autoimmune responses.

Antibodies: Functions and Role

• Antibodies bind to specific antigens.
• They can block pathogen interactions with host cells, promote phagocytosis, and neutralize toxins.

Complement Proteins and Complement Activation

• The complement system is a group of innate immune proteins that enhance the overall immune system's ability to respond to pathogens.
• Classical, lectin, and alternative pathways activate complement through the combination and splitting reaction of various proteins leading to lysis and inflammation.

Epidemiology

• Epidemiology studies diseases patterns, causes, and effects in entire populations.
• Endemic, epidemic, and pandemic disease are characterized by the distribution of disease prevalence.
• Key parameters in disease transmission to consider include the type of contact, mode, and route, for example, direct or indirect contact via fomites, droplets, vectors, carriers or other vehicles like contaminated water or food.

Herd Immunity

• Herd immunity is the immunity of a human population to an infectious agent, due to a large proportion of immune individuals; those individuals with immunity protect the susceptible.
• Herd immunity is a critical aspect of disease prevention and control.

Countermeasures

• Countermeasures are methods to reduce or prevent infections.
• Filters, chlorination of water, and vaccines are examples.
• Herd immunity is maintained by widespread vaccination.

The T7 Expression System

• The T7 expression system is a bacterial expression system allowing the controlled and rapid production of a specific protein and is designed for quick protein expression, and manipulation.

Site-Directed Mutagenesis & Gene Disruption

• Using this technique, one can deliberately introduce a specific change in DNA sequence (e.g., a mutation), generating the desired product quickly and efficiently.
• This allows for studying the influence of various mutations and identifying specific functional components of a gene/protein.

Recombinant DNA Technology: Synthesis of Insulin

• Insulin can be synthesised via this technique utilizing prokaryotes (e.g. E coli) or eukaryotes (e.g. yeast).
• This method allows for the production of complex proteins, from a foreign gene.

Production of Transgenic Plants and Viruses

• Plants and animal virus genes can be inserted using binary vectors inside specific tissues.
• These methods enable generating plants and animal cells that expressed the gene of interest after the insertion process.