Inhibitors of Translation Translation inhibitors are mostly antibiotics that target bacterial ribosomes. Since bacteria rely heavily on protein synthesis , the
Inhibitors of Translation Translation inhibitors are mostly antibiotics that target bacterial ribosomes. Since bacteria rely heavily on protein synthesis , these drugs disrupt translation and prevent bacterial growth. Differences Between Prokaryotic and Eukaryotic Ribosomes - Prokaryotic ribosomes : 70S , composed of 30S (small subunit) and 50S (large subunit) . - Eukaryotic ribosomes : 80S , composed of 40S (small subunit) and 60S (large subunit) . Most antibiotics target the differences between bacterial (prokaryotic) and human (eukaryotic) ribosomes to avoid harming human cells. --- Differences Between Translation in Prokaryotes and Eukaryotes Categories of Translation Inhibitors (Antibiotics) 1. Aminoglycosides - Mechanism :Bind to both 30S and 50S ribosomal subunits . - Block codon recognition , preventing translation. - Cause misreading of mRNA , leading to defective proteins. - Examples : Streptomycin , Gentamicin , Kanamycin . - Toxicity & Side Effects : Ototoxicity (hearing loss) – damages the ear. - Nephrotoxicity (kidney damage) – inhibits prostaglandins (vasodilators). - Vestibular damage – causes dizziness. - Resistance Mechanisms :Ribosomal mutations preventing drug binding. - Enzymatic modification of the drug ( phosphorylation ). - Efflux pumps remove the drug from bacteria. 2. Tetracyclines - Mechanism :Bind to 30S ribosomal subunit . - Block tRNA binding to mRNA, preventing elongation. - Examples : Doxycycline , Oxytetracycline , Chlorotetracycline . - Toxicity & Side Effects : Photosensitivity – avoid sun exposure. - Teeth discoloration – should not be given to children . - Gastrointestinal distress . - Resistance Mechanisms : Efflux pumps remove the drug from bacterial cells. - Mutations in ribosomal binding sites. - Plasmid-coded resistance genes . 3. Macrolides - Mechanism :Bind to 50S ribosomal subunit . - Prevent mRNA movement between the A-site and P-site , stopping elongation. - Examples : Erythromycin , Clarithromycin , Azithromycin . - Toxicity & Side Effects : Gastrointestinal (GIT) disturbances . - Metallic taste . - Resistance Mechanisms : Methylation of 50S ribosomal subunit , preventing drug binding. - Efflux pumps remove the drug from the cell (plasmid-mediated). 4. Chloramphenicol - Mechanism :Binds to 50S ribosomal subunit . - Inhibits peptide bond formation , stopping protein synthesis. - Toxicity & Side Effects : Bone marrow suppression – can lead to aplastic anemia . - Mitochondrial damage – causes toxicity in human cells. - Resistance Mechanisms :Bacteria produce enzymes that modify chloramphenicol , preventing its action. Short-Answer Questions and Answers 1. Discuss in detail the mechanisms, actions, toxicity, and effects of translation-inhibiting drugs. Answer: - Aminoglycosides bind both ribosomal subunits , interfere with codon recognition , and cause misreading of mRNA . They are bactericidal but cause renal and ear toxicity . - Tetracyclines bind to 30S , block tRNA attachment , and prevent elongation . They cause teeth discoloration and photosensitivity . - Macrolides bind 50S , block mRNA movement , and prevent elongation . They cause GIT issues and metallic taste . - Chloramphenicol binds 50S , blocks peptide bond formation , but has high toxicity (anemia, mitochondrial damage) . 2. Differentiate between translation in prokaryotes and eukaryotes. (10 marks) Answer: 3. Differentiate between translation and transcription. (10 marks) Answer: 10 key points differentiating transcription and translation : Transcription vs. Translation (10 Points) Recombinant DNA Technology & Cloning Recombinant DNA technology, also known as genetic engineering , involves modifying genetic material to create new combinations of DNA. This technology was developed in the 1970s after the discovery of restriction enzymes in bacteria. Key Components of Recombinant DNA Technology - Restriction Enzymes Found in bacteria. - Function: Cut DNA at specific restriction sites (also called palindromic sequences ). - Example: EcoRI (from Escherichia coli) recognizes 5' AATTC 3' and cuts at this site. - Produces either: Sticky ends (cohesive, easier for DNA recombination). - Blunt ends (straight cuts). - Plasmids (Vectors) Small, circular, extra-chromosomal DNA in bacteria. - Self-replicating, used to carry foreign DNA into host cells. - Contains: Origin of replication (Ori) – ensures self-replication. - Antibiotic resistance gene – helps in selecting transformed cells. - Multiple cloning site (MCS) – region with multiple restriction sites. - Vectors for Cloning Bacterial plasmids (circular, commonly used). - Bacteriophage vectors (linear DNA). - Ti plasmids (used for plant genetic modification). Steps in Recombinant DNA Technology 1. Identifying the Gene of Interest - Example: Insulin gene (used to treat Diabetes Mellitus Type 1 ). - Extracted from human DNA and cut using restriction enzymes . 2. Cutting & Preparing the Plasmid - Plasmid DNA is cut using the same restriction enzyme as the gene of interest. - Linearizes the plasmid, creating compatible sticky ends . 3. Inserting the Gene into the Plasmid - DNA Ligase enzyme joins the gene into the plasmid. - Forms a recombinant plasmid . - Example: Lac Z gene disruption confirms successful insertion. 4. Transferring the Recombinant Plasmid into Host Bacteria - The plasmid is introduced into E. coli (commonly JN 50g strain, which is non-virulent). - Methods of Gene Transfer : Physical Methods Electroporation – electrical shock creates pores for DNA entry. - Gene gun – shoots DNA-coated particles into cells. - Microinjection – injects DNA directly into cells. - Chemical Methods Calcium chloride treatment – makes bacterial membrane more permeable. - Biological Methods Liposomes – DNA enclosed in lipid vesicles enters cells. 5. Selecting & Growing Transformed Bacteria - The bacteria replicate the recombinant plasmid . - Cells with successful transformation express the gene of interest (e.g., insulin production). - Antibiotic resistance markers help in selecting transformed bacteria. Applications of Recombinant DNA Technology - Medical Applications Synthetic insulin (for diabetes treatment). - Growth hormones . - Gene therapy (correcting genetic disorders). - Agricultural Applications Genetically modified crops (GMOs) . - Pest-resistant plants (Bt cotton). - Industrial Applications Enzyme production (e.g., amylase for biofuel). - Biodegradable plastics . Selection of Recombinant Bacteria Using X-Gal and Streptomycin Once the recombinant plasmid is successfully introduced into bacteria, the next step is identifying which bacteria have successfully incorporated the desired gene. This is done using a selection medium containing X-Gal and streptomycin . 1. Selection Medium - The culture medium contains: X-Gal – a modified form of lactose used for selection. - Streptomycin – an antibiotic that prevents the growth of non-resistant bacteria. - Only bacteria with the recombinant plasmid (which contains a streptomycin resistance gene ) will survive in the medium. 2. The Role of X-Gal in Selection - X-Gal is used as an indicator to differentiate between transformed (recombinant) and non-transformed bacteria based on color changes . - The selection is based on LacZ gene activity , which encodes the enzyme β-galactosidase . - When X-Gal is broken down by β-galactosidase, it produces a blue color . 3. Interpretation of Results (Blue-White Screening) After incubating the culture at 37°C for 16 hours , the bacterial colonies appear in two colors : - Blue colonies :Contain a non-disrupted LacZ gene . - Produce β-galactosidase , breaking down X-Gal into a blue pigment. - Do not contain the inserted gene (e.g., insulin gene). - White colonies : Recombinant bacteria where the LacZ gene was interrupted by the inserted gene. - Cannot break down X-Gal , so they appear white . - These colonies contain the gene of interest (e.g., insulin gene). 4. Reculturing Recombinant Bacteria - White colonies (recombinant bacteri