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Download L3 - DNA Translation (Protein Synthesis
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The Central Dogma Continued: Translation (Protein Synthesis) Translation • The translation of the information contained in mRNA into protein requires a supply of amino acids, tRNA molecules, mRNA, ribosomes, and a number of enzymes. • Translation occurs in four steps: 1. 2. 3. 4. Translation, cont. Activation of tRNA Initiation Elongation Termination Translation, cont. Activation Initiation • In translation, tRNA molecules carry amino acids to mRNA bound to ribosomes. A tRNA molecule carrying its specified amino acid is termed an activated tRNA. • The initiation of protein synthesis begins as a tRNA molecule carrying the N-terminal amino acid (usually a form of methionine) meets with the mRNA molecule on a ribosome forming a complex at a specific site called the peptidyl site, or P site. • Activation occurs by the reaction of tRNA molecules with ATP. Once activated, enzymes in the cell match the correct amino acid with the correct tRNA molecule. • Each cell contains a complete set of enzymes for all of the AA/tRNA combinations. Translation, cont. Structure of tRNA: • The amino acid attachment site is at the open end of the cloverleaf (the 3’ end), and the anticodon is located in the loop opposite the open end. • The P site represents the initiation site on a ribosome where the initiation codon on the mRNA must match up with the anticodon on the tRNA. The binding occurs as H-bonds are formed between the bases of the mRNA codon and the anticodon on the tRNA meet. Translation, cont. Initiation • To form the initiated complex, mRNA and a small ribosomal subunit join so the initiating codon (AUG) is aligned with P site of subunit. • tRNA brings in methionine (eukaryotes) or Nformylmethionine (prokaryotes). • Large ribosomal subunit attaches to complete ribosome. 1 Translation, cont. Elongation • The next incoming tRNA bonds at a second binding site called the A site on the mRNA. • Once the tRNA binds at the A site with the correct amino acid, a peptide bond is formed between the amino acids attached to the 3’ end of the two tRNA molecules catalyzed by peptidyl transferase. • The ribosome then moves along the mRNA molecule to the next binding codon, known as translocation, and releases the “empty” tRNA from the p site as the tRNA from the A site move in to make the A site available to receive the next tRNA. Translation, cont. Elongation Translation, cont. Translation, cont. Termination • Once the ribosome reaches a termination codon, termination begins • The now empty ribosome then dissociates and can then bind to another strand of mRNA to begin again. • Several ribosomes can be attached to an mRNA strand at any one time resulting in the formation of many peptide chains at the same time. Such complexes of many ribosomes on one mRNA molecules are called polysomes or polyribosomes. Mutations A mutation is any change resulting in an incorrect base sequence on DNA. Occur naturally during DNA replication. Induced by environmental factors. • Ionizing radiation (X-rays, UV, γ) • Mutagens – chemical agents. Not necessarily harmful. Could lead to the production of beneficial, harmful or inactive proteins. Rough Endoplasmic Reticulum with Riobosomes along outer surface. Ribosomes along two long mRNA Molecules. Upper: emerging Nascent Polypeptides. They emerge from each ribosome during translation Genetic Engineering Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that are applied to the direct manipulation of an organism's genes. Genetic engineering uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Since a protein sequence is specified by a segment of DNA called a gene, novel versions of that protein can be produced by changing the DNA sequence of the gene. 2 i.e. Recombinant DNA Genetic Engineering There are a number of ways through which genetic engineering is accomplished. Essentially, the process has four main steps: 1.Isolation of the genes of interest. 2.Insertion of the genes into a transfer vector. 3.Replication of cellular genome for production of modified gene. 4.Separation of the genetically modified organism or protein of interest. Bacteria plasmids are often used as vectors Recombinant DNA Con’t. Genetically modified bacterium can then be separated from unmodified bacterium and reproduced to harvest products of modified DNA. VIDEO 3