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?
The Coding Problem • 1-1 correspondence – NO – 4 possibilities • 2-1 correspondence – NO – 16 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
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
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c ^ ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c Reading Frames & Mutation Types • Frame shift mutations • Original reading frame is frame a • Insertions or deletions shift the reading frame
ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW 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. ^ 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 • 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 …
Special codons: • AUG (which specifies methionine) = start codon • AUG specifies internal methionines also • UAA, UAG and UGA = termination, or stop, codons • The code isdegenerate • 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 thewobble base • The code isnearly universal • Only a few rare exceptions have been noted