The Genetic Code and Transcription Lecture PDF

The Genetic Code and Transcription Biochemical and Physiological Genetics Level 2 / 1st semester 2020  Genetic Code  Genetic information is stored in DNA by means of a triplet code that is nearly universal to all living things on Earth.  The genetic code i...

The Genetic Code and Transcription Biochemical and Physiological Genetics Level 2 / 1st semester 2020  Genetic Code  Genetic information is stored in DNA by means of a triplet code that is nearly universal to all living things on Earth.  The genetic code is initially transferred from DNA to RNA, in the process of transcription.  Once transferred to RNA, the genetic code exists as triplet codons, which are sets of three nucleotides in which each nucleotide is one of the four kinds of ribonucleotides composing RNA.  Genetic Code  RNA’s four ribonucleotides, analogous to an alphabet of four “letters,” can be arranged into 64 different three-letter sequences. Most of the triplets in RNA encode one of the 20 amino acids present in proteins, which are the end products of most genes.  Several codons act as signals that initiate or terminate protein synthesis. The Genetic Code Uses Ribonucleotide Bases as “Letters”   the general features that characterize it: 1- The genetic code is written in linear form, using as “letters” the ribonucleotide bases that compose mRNA molecules. 2- Each “word” within the mRNA consists of three ribonucleotide letters, thus referred to as a triplet code. 3- The code is unambiguous each triplet specifies only a single amino acid. 4- The code contains one “start” and three “stop” signals, triplets that initiate and terminate translation, respectively  The Genetic Code Flowchart illustrating how genetic information encoded in DNA produces protein. Early Studies Established the Basic Operational Patterns of the Code  The Triplet Nature of the Code effect of frameshift mutations on a DNA sequence repeating the triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplets out of the reading frame. (b) The insertion of three nucleotides changes only two triplets, after which the original reading frame is reestablished. The Nonoverlapping Nature of the Code The Commaless and Degenerate Nature of the Code  Studies by Nirenberg, Matthaei, and Others Led to Deciphering of the Code In 1961, Marshall Nirenberg and J. Heinrich Matthaei became the first to characterize specific coding sequences, laying a cornerstone for the complete analysis of the genetic code. Their success, as well as that of others who made important contributions in deciphering the code, was dependent on the use of two experimental tools, an in vitro (cell-free in a test tube) protein-synthesizing system and the enzyme polynucleotide phosphorylase, which allowed the production of synthetic mRNAs. These mRNAs served as templates for polypeptide synthesis in the cell-free system.  Synthesizing Polypeptides in a Cell-Free System In the cell-free protein-synthesizing system, amino acids are incorporated into polypeptide chains. The process begins with an in vitro mixture containing all the essential factors for protein synthesis in the cell: ribosomes, tRNAs, amino acids, and other molecules essential to translation. To allow scientists to follow (or trace) the progress of protein synthesis, one or more of the amino acids must be radioactive. Finally, an mRNA must be added, to serve as the template to be translated.  Synthesizing Polypeptides in a Cell-Free System The reaction catalyzed by the enzyme polynucleotide phosphorylase. Note that the equilibrium of the reaction favors the degradation of RNA but that the reaction can be “forced” in the direction favoring synthesis.  Homopolymer Codes For their initial experiments, Nirenberg and Matthaei synthesized RNA homopolymers, RNA molecules containing only one type of ribonucleotide, and used them for synthesizing polypeptides in vitro. In other words, the mRNA they used in their cell-free protein-synthesizing system was either UUUUUU... , AAAAAA... , CCCCCC... , or GGGGGG.... They tested each of these types of mRNA to see which, if any, amino acids were consequently incorporated into newly synthesized proteins. Homopolymer Codes Mixed Copolymers Mixed Copolymers Results and interpretation of a mixed copolymer experiment in which a ratio of 1A:5C is used (1/6A:5/6C).  The Triplet Binding Assay The behavior of the components during the triplet-binding assay. When the UUU triplet is positioned in the ribosome, it acts as a codon, attracting the complementary AAA anticodon of the charged tRNAphe.  The Triplet Binding Assay This technique took advantage of the observation that ribosomes, when presented in vitro with an RNA sequence as short as three ribonucleotides, will bind to it and form a complex similar to what is found in vivo. The triplet RNA sequence acts like a codon in mRNA, attracting a tRNA molecule containing a complementary sequence. Such a triplet sequence in tRNA, that is, complementary to a codon of mRNA, is known as an anticodon. The Triplet Binding Assay Repeating Copolymers The conversion of di-, tri-, and tetranucleotides into repeating copolymers. The triplet codons produced in each case are shown. Repeating Copolymers The Coding Dictionary Reveals Several Interesting Patterns among the 64 Codons  Degeneracy and the Wobble Hypothesis The coding dictionary. AUG encodes methionine, which initiates most polypeptide chains. All other amino acids except tryptophan, which is encoded only by UGG, are represented by two to six triplets. The triplets UAA, UAG, and UGA are termination signals and do not encode any amino acids. Degeneracy and the Wobble Hypothesis