The Genetic Code 肖航 200431060029

Do you know these people? Robert Holley, the discoverer of the transfer RNA - tRNA. George Gamow, Russian physicist, founded the "RNA Tie Club" in 1954. Har Gobind Khorana, creator of new methods to produce synthetic nucleic acids. arshall Nirenberg, the scientist that deciphered the genetic code in 1961.

Preview The translation of genetic and is mediated by special adaptor molecules knows as transfer RNAs(tRNAs). Three consecutive nucleotides are known as codons. With four possible nucleotides at each position, the total number of permutations of there triplets is 64. The DNA molecule, the carrier of the genetic information. RNA, a molecule which resembles DNA, is however single-stranded.

OutLine The Code Is Degenerate Three Rules Govern the Genetic Code Suppressor Mutations Can Reside in the Same or a Different Gene The Code is Nearly Universal

The Code Is Degenerate(简并性) Many amino acides are specified by more than one codon, the phenomenon called degeneracy. When the first two nucletides are identical, the third nucleotide can be either cytosine or uracil and the codon will still code for the same amino acide Often,adenine and guanine are similarly interchangeable UUU、UUC phenylalanine UCU、UCC、UCA、UCG、AGU、AGC serine

The Code Is Degenerate(简并性) Not all degeneracy is based on equivalence of the first two nucleotides There can be great variation in the AT/GC ratios in the DNA of various organisms without correspondingly large changes in the relative proportion of amino acids in the proteins UUA、UUG、CUU、CUC、CUA、CUG leucine

The Genetic Code is Unambiguous In general, no codon specifies more than one amino acid. The exceptions so far are AUG, UGA and UAG. In the first case, AUG specifies both Methionine and N-formyl-Methionine, which is used to initiate protein synthesis in bacteria. In the second case, UGA specifies the twenty-first amino-acid selenocysteine as well as being a stop codon. And, in the last case, UAG specifies the twenty second amino acid (the most recent to be added to the list), pyrrolysine.

Perceiving Order in the Makeup of the Code The code evolved in such a way as to minimize the deleterious effects of mutations Mutation in the first position of a codon will often give a similar amino acid. Codons with pyrimidines in the second position specify mostly hydrophobic amino acids,with purines in the second position correspond mostly to polar amino acids. Change in the third position rarely will a different amino acid be specified,even transversion.

Perceiving Order in the Makeup of the Code Whenever the first two positions of a codon are both occupied by G or C, each of the four nucleotides in the third position specifies the same amino acid. Whenever the first two positions of the codon are both occupied by A or U, the identity of the third nucleotide does make a difference.

Wobble in the Anticodon The base at the 5’ end of the anticodon is no as spatically confined as the other two allowing it to form hydrogen bonds with any of several based located at the 3’ end of a codon. Base in Anticodon Base in Codon G C A U I U or C A or G A,U,or C Paring Combinations with the Wobble Concept

Wobble in the Anticodon The wobble rules do not permit any single tRNA molecule to recognize four different codons. Question:why the wobble is in the 3’ position of the codon?

Wobble in the Anticodon The three anticodon base all point in roughly the same direction, with their exact conformations largely determined by stackinginteractions between the flat surfaces of the bases.The first (5’) anticodon base is at the end of the stack and is perhaps less restricted in its movements than the other two anticodon bases.

Three Codons Direct Chain Termination UAA,UAG,UGA are read not by special tRNA, but by specific proteins known as release factors(RF1 and RF2 in bacteria and eRF1 in eukaryotes). Release factors enter the A site of the ribosome and trigger hydrolysis of the peptidyl-tRNA occupying the P site, resulting in the release of the newlysynthesized protein.

How the Code Was Cracked The first steps to solving the Genetic Code depended on the development of a cell-free in vitro translation system by Paul Zamecnik (right). This system which consisted of a membrane-free cell supernatent, ATP, GTP, radioactively labelled amino-acids and RNA, was capable of directing the synthesis of radioactively labelled protein.

Stimulation of Amino Acid Incorporation by Synthetic mRNAs The dependence of cell extracts on externally added mRNA provided an opportunity to elucidate the nature of the code using synthetic polyribonucleotides. These synthetic templates were created using the enzyme polynucleotide phosphorylase,which catalyzes the reaction: [XMP]n + XDP [XMP] n+1 + Polynucleotide phosphorylase is normally responsible for breaking down RNA.Howere, by use of high nucleoside diphosphate concentrations this enzyme can be made to catalyze the formation of internucleotide 3’ 5’ phosphodiester bonds and thus make RNA molecules. Addition of two or more different diphosphates produces mixed copolymers such as poly-AU poly-AC poly-CU and poly-AGCU. P

Stimulation of Amino Acid Incorporation by Synthetic mRNAs The figure shows the reversible reactions of synthesis or degradation of polyadenylic acid catalyzed by the enzyme polynucleotide phosphorylase

Poly-U Code for Polyphenylalanine A high magnesium concentration circumvents the need for initiation factors and the special initiator fMet-tRNAm allowing chain initiation to take place without the proper signals in the mRNA. Poly-U selects phenylalanyl tRNA molecules exclusively , thereby forming a polypeptide chain containing only pheny-lalanine. On the basis of analogous experiments with poly-C and poly-A,CCC was assigned as a proline codon and AAA as a lysine codon. The guanine residues in poly-G firmly hydrogen bond to each other and form multistranded triple helicase that do not bind to ribosomes. The experiment which used uracil (U) as a template produced a protein entirely made up of the amino acid phenylalanine (F). The first letter of the genetic code was hence identified.

Mixed Copolymers Allowed Additional Codon Assignments This use of homopolymers is clearly quite limited. The use of random mixed copolymers helped to extend the utility of the system and the information obtained from it. Random copolymers can be synthesized from a mixture of two ribonucleotides with polynucleotide phosphorylase. Thus if ADP and CDP are used in a 5:1 ratio, then the frequency of each possible triplet in the synthesized RNA will vary according to this ratio. For example, AAA triplets will be found 100 times more frequently than CCC triplets.

Transfer DNA Binding to Defined Trinucleotide Codons aminoacylated tRNAs could be bound to ribosomes if the ribosomes contained trinucleotides acting as mRNA.

Codon Assignments from Repeating Copolymers tri- and tetra-nucleotides could be polymerized into polymers with repeating sequences that could be used in cell-free in vitro translation assays . In the case of trinucleotides, three polypeptides will be synthesized, each of which is a homopolymer of a single amino acid.

Three Rues Govern The Genetic Code Codons are read in a 5’ to 3’ direction. Codons are nonoverlapping and the message contains no gaps. The message is translated in a fixed reading frame, which is set by the initiation codon.

Three Kinds of Point Mutations Alter the Genetic Code missense mutation:an alteration that changes a condon specific for one amino acid to a codon specific for another amino acid . nonsense/stop mutation: an alteration causing a change to a chain-termination codon. Frameshift mutation: insertions or deletions of one or a smal number of base pairs that alter the reading frame.

Genetic Proof that the Code is Read in Units of Three 5’-GCU GCU AGC UGC AUG CUG CAU GCU GCU GCU-3’ Ala Ala Ser Cys Met Leu His Ala Ala Ala The finding indicated that the overall coding capacity of the gene had been chiefly left unaltered despite the presence of three mutations, each of which alone, or any two of which alone, would have drastically altered the reading frame of the gene’s message. Because the gene could tolerate three insertions but not one or two, the genetic code must be read in units of three.

Suppressor Mutations can Reside in The Same or a Different Gen Intragenic suppression intergenic occurring within the same gene as the original mutation,but at a different site occurring in another gene Suppressor genes:genes that cause suppression of mutations in other genes. One example of intragenic supression is missense mutation.The effect can sometime be reversed through an additional missense mutation in the same gene Another example of intragenic: frameshift mutation.

Suppressor Mutations can Reside in The Same or a Different Gen A deletion in the nucleotide coding sequence can result in an incomplete, inactive poly peptide chain. (b) The effect of the deletion, shown in panel a, can be overcome by a second mutation, an insertion in the coding sequence. This insertion results in the production of a complete polypeptide chain having two amino acid replacements. Depending on the change in sequence, the protein may have partial or full activity. Suppression of frameshift

Intergenic Suppression Involves Mutant tRNAs Suppressor genes do not act by changing the nucleotide sequence of a mutant gen.Instead, they change the way the mRNA template is read. nonsense mutations :A mutation in the anticodon of tRNA that alters the anticodon so it is now complementary to a nonsense codon allowing the tRNA to insert its cognate amino acid at this nonsense codon during translation. If a mutation occurs in the DNA that changes the AAG codon in the mRNA to UAG, the UAG codon will be read as a stop signal and the translation product will be a truncated (short) usually nonfunctional polypeptide.

Nonsense Suppressors also Read Normal Termination Signals The act of nonsense suppression can be viewed as a competition between the suppressor tRNA and the release factor. When a stop codon comes into the ribosomal A site , either read-through or polypeptidechain termination will occur, depending on which arrives first.

Proving the Vality of the Genetic Code A classic and instructive experiment in 1966 helped to validate the genetic code.

The Code is Nearly Universal The results of large-scale sequencing of genomes have confirmed the universality of the genetic code. Benefits of the universal codes: 1. Allow us to directly compare the protein coding sequences among all organisms. 2. Make it possible to express cloned copies of genes encoding useful protein in different host organism. Example: Human insulin ecpression in bacteria)

The Code is Nearly Universal However, in certain subcellular organelles, the genetic code is slightly different from the standard code. Mitochondrial tRNAs are unusual in the way that they decode mitochondrial messages. Only 22 tRNAs are present in mammalian mitochondria. The U in the 5’ wobble position of a tRNA is capable of recognizing all four bases in the 3’ of the codon.

The Code is Nearly Universal

The Code is Nearly Universal