Ribosomes PDF

CELL BIOLOGY Dr. Madani S. 2023/2024 RIBOSOMES Ribosomes Ribosomes are small, globular, non-membranous organelles found in prokaryotic and eukaryotic cells. They are involved in protein synthesis, the process by which messenger RNA (mRNA) is translated into amino acids, forming a polypeptide cha...

CELL BIOLOGY Dr. Madani S. 2023/2024 RIBOSOMES Ribosomes Ribosomes are small, globular, non-membranous organelles found in prokaryotic and eukaryotic cells. They are involved in protein synthesis, the process by which messenger RNA (mRNA) is translated into amino acids, forming a polypeptide chain. Ribosomes are composed of two major subunits: a small subunit that is responsible for decoding mRNA, and a large subunit that is responsible for catalyzing peptide bond formation. These subunits are made up of a combination of 65% ribosomal RNA (rRNA) and 35% ribosomal proteins. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes under the Transmission electron microscope Under the transmission electron microscope, using the negative contrast technique, ribosomes appear as distinct, globular, electron-dense particles ranging from 14 to 23 nm in diameter. They can be found in the cytosol, either as two separate subunits when they are inactive or joined in chains called polysomes (polyribosomes) when they are actively building proteins. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes under the Transmission electron microscope In eukaryotic cells, ribosomes can also be attached to the cytosolic face of the rough endoplasmic reticulum and the outer nuclear membrane. They are also found in mitochondria and chloroplasts. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes under the Transmission electron microscope Micrograph of free ribosomes under the Micrograph of polyribosomes under the Micrograph of ribosomes attached to the Transmission electron microscope Transmission electron microscope RER and the outer nuclear membrane under the Transmission electron microscope CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes isolation and analysis Ribosomes are isolated using the density gradient ultracentrifugation. Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact that, in suspension, particles that are more dense than the solvent will sediment, while those that are less dense will float. Sedimentation is the process by which particles settle out of a fluid under the influence of gravity. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes isolation and analysis Isolating ribosomes using differential centrifugation is a multi-step process that separates ribosomes from other cellular components: 1. Cell collection: Obtain cells from either a cell culture or a tissue sample. 2. Cell homogenization: Break down the cells into a homogenate using methods like sonication or grinding. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes isolation and analysis 3. Differential centrifugation: Subject the homogenate to centrifugation at increasing speeds. ▪ Pellet 3: Contains microsomes, small rough vesicles derived from the rough endoplasmic reticulum. ▪ Pellet 4: Contains free ribosomes. 3. Sucrose gradient centrifugation: Characterize ribosomes and ribosomal subunits based on their sedimentation coefficient (Svedberg units) using a sucrose concentration gradient. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes isolation and analysis Ribosomes sites The ribosome's binding sites play crucial roles in the translation process. 1. mRNA Binding Site: Located on the 18S rRNA of the small subunit in eukaryotes (16S rRNA in prokaryotes), this site binds to the mRNA molecule, ensuring proper positioning for translation. 2. Aminoacyl-tRNA Binding Site (A site): Found on the 28S rRNA of the large subunit in eukaryotes (23S rRNA in prokaryotes), this site binds to the tRNA carrying the incoming amino acid, ready to be added to the growing polypeptide chain. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes sites 3. Peptidyl-tRNA Binding Site (P site): located on the 28S rRNA of the large subunit in eukaryotes (23S rRNA in prokaryotes), the P site binds to the tRNA carrying the growing polypeptide chain. It's here that a new peptide bond is formed between the incoming amino acid and the growing polypeptide chain. 4. Exit Site (E site): Situated on the 28S rRNA of the large subunit in eukaryotes (23S rRNA in prokaryotes), the E site serves as the exit point for the tRNA that has delivered its amino acid. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes function: protein translation Ribosomes play a crucial role in protein translation according to the following steps : 1. Initiation: The small subunit binds to the mRNA molecule marking the start of translation by the codon AUG. 2. Elongation: The ribosome moves along the mRNA, reading the codon triplets (three-nucleotide sequences) one by one. 3. Amino Acid Matching: At each codon, the small subunit matches a complementary anticodon carried by a tRNA molecule. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes function: protein translation 4. Peptide Bond Formation: The large subunit catalyzes the formation of a peptide bond between the growing polypeptide chain and the amino acid carried by the tRNA. 5. Translocation: The ribosome shifts along the mRNA, bringing the next codon into the decoding site and the tRNA in the P site to the exit site. 6. Termination: When a stop codon UAA is encountered, the ribosome releases the completed polypeptide chain and disassembles. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes function: protein translation CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024 Ribosomes function: protein translation Ribosome Components roles in protein translation: Small subunit: Decodes the mRNA sequence and selects the appropriate tRNAs. Large subunit: Catalyzes the formation of peptide bonds and anchors the ribosome to the mRNA. mRNA: Carries the genetic information that is translated into a protein sequence. tRNA: Serves as an adapter molecule, carrying specific amino acids to the ribosome based on the mRNA codons. CELL BIOLOGY/ 1st Academic year / Dr. MADANI S. /2023-2024