Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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. coli tryptophan synthase

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Elucidating the Genetic Code A triplet code is required: 4 3 = 64, but 4 2 = 16 - not enough for 20 amino acids But is the code overlapping? See Figure 32.2 And is the code punctuated?

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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?

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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  2  2 Subunits from 334 to more than 1000 residues Two different mechanisms (See Figure 32.5)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 on specificity of several aminoacyl-tRNA synthetases

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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"

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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