Inhibitors of Transcription Transcription inhibitors are compounds that interfere with RNA synthesis by targeting RNA polymerase. Some of these inhibitors are
Inhibitors of Transcription Transcription inhibitors are compounds that interfere with RNA synthesis by targeting RNA polymerase. Some of these inhibitors are used as antibiotics , while others are natural toxins. 1. Rifamycin (Rifampin) – Inhibitor of Prokaryotic Transcription - Produced by : Amycolatopsis rifamycinica (a bacterial species). - Targets : Prokaryotic RNA polymerase . - Mechanism of Action :Binds to the β-subunit of RNA polymerase . - Prevents elongation of the mRNA strand. - Inhibits transcription initiation. - Clinical Use :Used to treat Mycobacterium tuberculosis (causes tuberculosis ). - Effective against other bacterial infections. 2. α-Amanitin – Inhibitor of Eukaryotic Transcription - Produced by : Amanita phalloides (death cap mushroom). - Targets : Eukaryotic RNA polymerase II . - Mechanism of Action : Strongly inhibits RNA polymerase II , which is responsible for synthesizing mRNA . - Moderate effect on RNA polymerase III (tRNA synthesis). - No effect on RNA polymerase I (rRNA synthesis). - Effects :Leads to severe hepatotoxicity (liver damage) . - Symptoms include nausea, vomiting, diarrhea , and eventually organ failure . Differences Between Transcription in Prokaryotes and Eukaryotes Post-Transcriptional Modifications in Eukaryotic mRNA - 5’ Capping :Addition of a 7-methylguanosine cap at the 5’ end. - Protects mRNA from degradation and helps in ribosome binding . - Polyadenylation :Addition of a poly-A tail at the 3’ end (about 200 adenine residues). - Helps in stability and nuclear export of mRNA. - Splicing :Removal of introns (non-coding sequences). - Exons (coding regions) are joined together to form mature mRNA . Summary - Rifamycin inhibits prokaryotic transcription by binding to RNA polymerase β-subunit . - α-Amanitin inhibits eukaryotic transcription by targeting RNA polymerase II . - Key differences exist between transcription in prokaryotes and eukaryotes, including location, mRNA processing, regulation, and termination. - Eukaryotic mRNA requires post-transcriptional modifications before translation. Translation: Protein Biosynthesis Translation is the second step in gene expression , following transcription. It is the process by which ribosomes synthesize proteins using mRNA as a template. 1. Location of Translation - Prokaryotes : Occurs in free ribosomes in the cytoplasm . - Eukaryotes : Ribosomes can be free in the cytoplasm or attached to the rough endoplasmic reticulum (RER) , where protein processing occurs . 2. Stages of Translation Protein synthesis occurs in three major steps : (A) Initiation - The goal is to bring together the mRNA , tRNA carrying the first amino acid , and the ribosomal subunits . - The mRNA contains a sequence of codons (triplets of nucleotides) that dictate which amino acids will be incorporated into the protein. - tRNA molecules carry amino acids to the ribosome. - The ribosome assembles , with:The small ribosomal subunit binding to the mRNA . - The large ribosomal subunit joining later to complete the ribosome. (B) Elongation - The ribosome moves along the mRNA , reading codons and adding amino acids in the correct sequence. - Each new amino acid is added to the growing protein chain by peptide bond formation . - Three key sites in the ribosome: A (Aminoacyl) Site – Binds incoming tRNA carrying an amino acid. - P (Peptidyl) Site – Holds tRNA with the growing polypeptide chain . - E (Exit) Site – Where empty tRNA exits after donating its amino acid. (C) Termination - Occurs when the ribosome reaches a stop codon ( UAA, UAG, UGA ) on the mRNA. - A release factor binds to the ribosome, causing it to disassemble , and the newly formed protein is released . 3. Role of tRNA in Translation - tRNA (Transfer RNA) molecules bring amino acids from the cytoplasm to the ribosome . - tRNA has a unique cloverleaf structure with 73-93 nucleotides . - Four distinct regions (lobes) are present: Anticodon Loop – Contains a sequence complementary to the mRNA codon to ensure correct amino acid pairing. - Amino Acid Attachment Site – Located at the 3' end , where the correct amino acid binds. 4. Summary of Key Concepts - Translation is the process of protein synthesis from mRNA. - Takes place in the ribosomes in both prokaryotes and eukaryotes. - Involves three stages : Initiation, Elongation, and Termination . - tRNA molecules play a crucial role by bringing amino acids to the ribosome and matching codons with their anticodons. Aminoacylation of tRNA (tRNA Charging) 1. Attachment of Amino Acids to tRNA - Each tRNA molecule must be attached to its corresponding amino acid to participate in protein synthesis. - This attachment process is catalyzed by an enzyme called aminoacyl-tRNA synthetase . - There are 20 standard amino acids involved in protein synthesis. - Each amino acid has a specific synthetase enzyme that facilitates its attachment to tRNA. 2. Naming of Aminoacyl-tRNA Synthetase - The naming of synthetase enzymes is based on the amino acid they attach:Example: The enzyme that attaches leucine to tRNA is called leucyl-tRNA synthetase . - Example: The enzyme that attaches glycine to tRNA is called glycyl-tRNA synthetase . 3. Types of tRNA Molecules - When an amino acid is correctly attached to its tRNA, the resulting molecule is called acylated tRNA or charged tRNA . - If a tRNA molecule lacks an amino acid (e.g., before aminoacylation), it is called uncharged tRNA . - If a tRNA molecule is mistakenly attached to the wrong amino acid, it is referred to as mischarged tRNA . 4. tRNA and Codon-Anticodon Interactions - Despite having only 20 amino acids , cells possess around 45 different tRNA molecules . - Messenger RNA ( mRNA ) contains 61 codons that specify amino acids. - The 45 different tRNA molecules must recognize 61 codons on mRNA, which is possible due to the "wobble hypothesis" . 5. The Wobble Hypothesis - Each tRNA molecule carries an anticodon , which pairs with a codon on the mRNA. - The third base of the codon (the "wobble position" ) can pair with more than one nucleotide , allowing fewer tRNA molecules to recognize multiple codons. - Example: At the wobble position , uracil (U) on the tRNA anticodon can pair with adenine (A) or guanine (G) in the mRNA codon. 6. Role of Modified Nucleotides - Some tRNA anticodons contain modified bases, such as inosine (I) , which can pair with uracil (U), adenine (A) , or cytosine ( C) on mRNA codons. - These modifications further increase the flexibility of codon-anticodon pairing. 7. Errors in tRNA Charging (Mischarged tRNA) - If a tRNA is mischarged (attached to the wrong amino acid), it must be corrected before translation. - Cells have proofreading mechanisms that prevent errors: Hydrolytic editing removes incorrectly attached amino acids from the tRNA. - If mischarged tRNA is not corrected, the incorrect amino acid could be incorporated into the protein, leading to malfunction. - If codon-anticodon binding does not match, translation is halted, preventing errors in protein synthesis. Steps in Transcription and Translation 1. Transcription Overview Transcription is the process of converting DNA into mRNA , which occurs in three main stages: - Initiation - Elongation - Termination Steps in Translation Translation is the process by which ribosomes read mRNA sequences and build proteins. It has three stages: A. Initiation (Starting Translation) The goal of initiation is to bring together: - mRNA - tRNA carrying the first amino acid - Two ribosomal subunits (small and large) Steps in Initiation - Binding of the Small Ribosomal Subunit to mRNA In prokaryotes , mRNA binds to the small ribosomal subunit at a unique sequence called the Shine-Dalgarno sequence . - In eukaryotes , the ribosome recognizes the 5' cap at the beginning of mRNA. Without this cap, mRNA cannot bind to the ribosome. - Binding of the Initiator tRNA A special initiator tRNA binds to the start codon (AUG) on the mRNA. - In prokaryotes , this tRNA carries N-formylmethionine (fMe