Translating the Genetic Code Gene expression part 3

An overview of gene expression Figure 13.2

The Idea of A Code 20 amino acids 4 nucleotides How do nucleic acids composed of 4 nucleotides specify the synthesis of proteins composed of 20 different amino acids? Figure: 12-01 Caption: Flow of genetic information encoded in DNA to messenger RNA to protein.

The Coding Problem 1-1 correspondence – NO – 4 possibilities 3-1 correspondence – YES – 64 possibilities

Determination of Triplet Nature of Code Aacridines & flavinoids cause single nucleotide deletions and insertions respectively Insertion of 1 base shifts frame & creates non-sense mutation Deletion of base in mutant restores frame (suppresses 1st mutation) Recombination between mutants showed that insertion of 1 or 2 bases or removal of 1 or 2 bases doesn't restore frame, but insertion of 3 or removal of 3 allows frame to continue Figure: 12-02ab Caption: The effect of frameshift mutations on a DNA sequence repeating the triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplet reading frames. (b) The insertion of three nucleotides changes only two triplets, but the frame of reading is then reestablished to the original sequence.

Crick’s Experiments to Determine Triplet Nature of Code

So.. The code is 3 letter words, but what about punctuation? GROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c There is a message, but one must start at the right place to read it Code written in three letter words - codon There are three reading frames, but only one gives an intelligible message – frame b NOW THE CAT SAW THE DOG BUT DID NOT RUN A start codon (NOW) and a stop codon (END) define the frame to use

Any frame is potentially the reading frame! ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c The actual reading frame is called the Open Reading Frame or ORF

Reading Frames & Mutation Types Frame shift mutations Original reading frame is frame a Insertions or deletions shift the reading frame ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c ^

Reading Frames & Mutations ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c Once a ribosome begins translation in a particular frame (a) it does not shift frames Therefore, if a mutation shifts the reading frame in the mRNA, the ribosome will read the wrong frame. ^ ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW a b c NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE W..

Deciphering the Code Each amino acid in a protein is specified by 3 nucleotides of codon Each codon specifies only ONE amimo acid There are 64 possible codons but only 20 amino acids Degeneracy An amino acid can be specified by multiple codons A given codon still only specifies only one amino acid

Deciphering the Code: Three Approaches Synthesis and translation of homopolymer RNA & ratio polymer RNA followed by amino acid analysis Synthesis and translation of di, tri and tetra nucleotide repetitive RNA polymers followed by amino acid analysis Triplet RNA-tRNA binding assay and amino acid analysis

Synthetic RNA Templates and In Vitro Translation Figure: 12-03 Caption: The reaction catalyzed by the enzyme polynucleotide phosphorylase. Note that the equilibrium of the reaction favors the degradation of RNA but can be “forced” in the direction favoring synthesis. Synthesis of homopolymeric RNAs UUUUUUUUUU, AAAAAAAAAA, CCCCCCCCC, & GGGGGGGGG When translated produced polypeptides poly phenylalanine (UUU), polyproline (CCC), polylysine (AAA), and polyglycine (GGG) Therefore 4 codons were determined UUU = phe CCC = pro AAA = lys GGG = gly

Decoding the Genetic Code The enzyme polynucleotide phosphorylase polymerizes ribonucleoside diphosphates (NDPs) into RNA It does not use a template, the order of nucleotides is random The nucleotide sequence is controlled by the relative abundance of NDPs For example, if 70% GDP and 30% UDP are mixed together, then … Radiolabeled Amino Acid Added (%) Amino Acid Incorporated Glycine (GGG + GGU, UGG, GUG) 49 (34 + 15) Valine (GGU, UGG, GUG, GUU, UUG, UGU) 21 (15 + 6) Tryptophan (UGG, GUG, GGU) 15 Cysteine (UUG, GUU, UGU) 6 Leucine (UUG, GUU, UGU) Phenylalanine (UUU) 3 Possible Codons Percentage in the Random Polymer GGG 0.7 x 0.7 x 0.7 = 0.34 = 34% GGU 0.7 x 0.7 x 0.3 = 0.15 = 15% GUU 0.7 x 0.3 x 0.3 = 0.06 = 6% UUU 0.3 x 0.3 x 0.3 = 0.03 = 3% UGG 0.3 x 0.7 x 0.7 = 0.15 = 15% UUG 0.3 x 0.3 x 0.7 = 0.06 = 6% UGU 0.3 x 0.7 x 0.3 = 0.06 = 6% GUG 0.7 x 0.3 x 0.7 = 0.15 = 15%

Polymers of Nucleotide Repeats

Trinucleotide-tRNA Binding Analysis Figure: 12-05 Caption: An example of the triplet binding assay. The UUU triplet acts as a codon, attracting the complementary tRNAphe anticodon AAA.

Figure: 12-07 Caption: 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.

The code is nearly universal Special codons: AUG (which specifies methionine) = start codon AUG specifies internal methionines also UAA, UAG and UGA = termination, or stop, codons The code is degenerate More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base It is sometime referred to as the wobble base The code is nearly universal Only a few rare exceptions have been noted