Chapter 32 The Genetic Code to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777
Outline • 32.1 Elucidating the Genetic Code • 32.2 The Nature of the Genetic Code • 32.3 The Second Genetic Code • 32.4 Codon-Anticodon Pairing, Third-Base Degeneracy and the Wobble Hypothesis • 32.5 Codon Usage • 32.6 Nonsense Suppression
Translating the Message How does the sequence of mRNA translate into the sequence of a protein? • What is the genetic code? • How do you translate the "four-letter code" of mRNA into the "20-letter code" of proteins? • And what are the mechanics like? There is no obvious chemical affinity between the purine and pyrimidine bases and the amino acids that make protein. • As a "way out" of this dilemma, Crick proposed "adapter molecules" - they are tRNAs!
The Collinearity of Gene and Protein Structures • Watson and Crick's structure for DNA, together with Sanger's demonstration that protein sequences were unique and specific, made it seem likely that DNA sequence specified protein sequence • Yanofsky provided better evidence in 1964: he showed that the relative distances between mutations in DNA were proportional to the distances between amino acid sunstitutions in E. colitryptophan synthase
Elucidating the Genetic Code • A triplet code is required: 43 = 64, but 42 = 16 - not enough for 20 amino acids • But is the code overlapping? • See Figure 32.2 • And is the code punctuated?
The Nature of the Genetic Code • A group of three bases codes for one amino acid • The code is not overlapping • The base sequence is read from a fixed starting point, with no punctuation • The code is degenerate (in most cases, each amino acid can be designated by any of several triplets
Biochemists Break the Code Assignment of "codons" to their respective amino acids was achieved by in vitro biochemistry • Marshall Nirenberg and Heinrich Matthaei showed that poly-U produced polyphenylalanine in a cell-free solution from E. coli • Poly-A gave polylysine • Poly-C gave polyproline • Poly-G gave polyglycine • But what of others?
Getting at the Rest of the Code • Work with nucleotide copolymers (poly (A,C), etc.), revealed some of the codes • But Marshall Nirenberg and Philip Leder cracked the entire code in 1964 • They showed that trinucleotides bound to ribosomes could direct the binding of specific aminoacyl-tRNAs (See Figure 31.6) • By using C-14 labelled amino acids with all the possible trinucleotide codes, they elucidated all 64 correspondences in the code (Table 32.3) • Read also about Khorana's experiment
Features of the Genetic Code • All the codons have meaning: 61 specify amino acids, and the other 3 are "nonsense" or "stop" codons • The code is unambiguous - only one amino acid is indicated by each of the 61 codons • The code is degenerate - except for Trp and Met, each amino acid is coded by two or more codons • Codons representing the same or similar amino acids are similar in sequence • 2nd base pyrimidine: usually nonpolar amino acid • 2nd base purine: usually polar or charged aa
AA Activation for Prot. Synth. The Aminoacyl-tRNA Synthetases • Codons are recognized by aminoacyl-tRNAs • Base pairing must allow the tRNA to bring its particular amino acid to the ribosome • But aminoacyl-tRNAs do something else: activate the amino acid for transfer to peptide • Aminoacyl-tRNA synthetases do the critical job - linking the right amino acid with "cognate" tRNA • Two levels of specificity - one in forming the aminoacyl adenylate and one in linking to tRNA
Aminoacyl-tRNA Synthetases Mechanism and specificity • Deacylase activity "edits" and hydrolyzes misacylated aminoacyl-tRNAs • Despite common function, the synthetases are a diverse collection of enzymes • Four different quaternary structures: , 2, 4 and 22 • Subunits from 334 to more than 1000 residues • Two different mechanisms (See Figure 32.5)
Recognition of tRNAs by the aminoacyl-tRNA synthetases • Anticodon region is not the only recognition site • The "inside of the L" and other regions of the tRNA molecule are also important • Read pages 1080-1082 on specificity of several aminoacyl-tRNA synthetases
Third-Base Degeneracy and the Wobble Hypothesis • Codon-anticodon pairing is the crucial feature of the "reading of the code" • But what accounts for "degeneracy": are there 61 different anticodons, or can you get by with fewer than 61, due to lack of specificity at the third position? • Crick's Wobble Hypothesis argues for the second possibility - the first base of the anticodon (which matches the 3rd base of the codon) is referred to as the "wobble position"
The Wobble Hypothesis • The first two bases of the codon make normal (canonical) H-bond pairs with the 2nd and 3rd bases of the anticodon • At the remaining position, less stringent rules apply and non-canonical pairing may occur • The rules: first base U can recognize A or G, first base G can recognize U or C, and first base I can recognize U, C or A (I comes from deamination of A) • Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis too