Translating the Genetic Code. DANILO V. ROGAYAN JR. Faculty, Department of Natural Sciences

Transcription

1 Translating the Genetic Code DANILO V. ROGAYAN JR. Faculty, Department of Natural Sciences

2 An overview of gene expression Figure 13.2

3 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?

4 The Coding Problem 1-1 correspondence NO 4 possibilities 2-1 correspondence NO 16 possibilities 3-1 correspondence YES 64 possibilities

5 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 1 st 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

6 Crick s Experiments to Determine Triplet Nature of Code

7 So.. The code is 3 letter words, but what a b c about punctuation? GROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW 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

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

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

10 a b c Reading Frames & Mutations ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW 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. a b c ^ ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE W..

11 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

12 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

13 Synthetic RNA Templates and In Vitro Translation 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

14 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 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% Radiolabeled Amino Acid Added Glycine (GGG + GGU, UGG, GUG) Valine (GGU, UGG, GUG, GUU, UUG, UGU) Tryptophan (UGG, GUG, GGU) Cysteine (UUG, GUU, UGU) Leucine (UUG, GUU, UGU) Phenylalanine (UUU) (%) Amino Acid Incorporated 49 ( ) 21 (15 + 6)

15 Polymers of Nucleotide Repeats

16 Trinucleotide-tRNA Binding Analysis

17 Transcription Fill in the complementary bases for the blank DNA strand given below:

18 Transcription Fill in the complementary bases for the blank DNA strand given below: mrna: AUG AAG GAU AAA AGA ACA GUG GCG UGA AA: met-lys-asp-lys-arg-thr-val-ala-stop

19 Sample DNA: T A C T G A T T C G A C C C C C A T C A G G A A A A T C mrna: A U G A C U A G C U G G G G G U A U U A C U U U U A G trna: U A C U G A U C G A C C C C C A U A A U G A A A A U C AA: MET THR SER TRP GLY TYR TYR PHE STOP

20 Problem 1. DNA: T A C C G C T C C G C C G T C G A C A A T A C C A C T mrna: trna: AA: 2. T A C T C T C C A A A A A A A T A C C A A C C A A C T mrna: trna: AA:

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22 Solution 1. DNA: T A C C G C T C C G C C G T C G A C A A T A C C A C T mrna: A U G-G C G -A G G -C G G- C A G -C U G -U U A -U G G -U G A trna: U A C- C G C- U C C- G C C- G U C- G A C- A A U- A C C- A C U AA: TYR-ARG-SER-ALA-VAL-ASP-ASN-THR-STOP 2. DNA: T A C T C T C C A A A A A A A T A C C A A C C A A C T mrna: A U G- A G A- G G U- U U U- U U U- A U G -G U U -G G U -U G A trna: U A C- U C U- C C A -A A A- A A A- U A C- C A A -C C A- A C U AA: TYR-SER-PRO-LYS-TRY-GLN-PRO-THR-STOP

23 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

24 Lakô hã salamát! Maraming salamat!