Chapter 22 (Part 1)

1. Chapter 22 (Part 1) Protein Synthesis

2. 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!

3. 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 substitutions in E. coli tryptophan synthase

4. Elucidating the Genetic Code • How does DNA code for 20 different amino acids? • 2 letter code would allow for only 16 possible combinations. • 4 letter code would allow for 256 possible combinations. • 3 letter code would allow for 64 different combinations • Is the code overlapping? • Is the code punctuated?

6. 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)

7. How the code was broken • 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?

8. 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 • By using C-14 labelled amino acids with all the possible trinucleotide codes, they elucidated all 64 correspondences in the code

10. 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 • First 2 codons of triplet are often enough to specify amino acid. Third position differs • Codons representing the same or similar amino acids are similar in sequence (Glu and Asp)

12. tRNAs • tRNAs are interpreters of the genetic code • Length = 73 – 95 bases • Have extensive 2o structure • Acceptor arm – position where amino acid attached • Anticodon – complementary to mRNA • Several covalently modified bases • Gray bases are conserved between tRNAs

13. tRNAs: 2o vs 3o Structure

14. Third-Base Degeneracy • 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"

15. The Wobble Hypothesis • The first two bases of the codon make normal 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

18. AA Activation for Prot. Synth. • 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

19. Aminoacyl-tRNA Synthetase Amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi • Most species have at least 20 different aminoacyl-tRNA synthetases. • Typically one enzyme is able to recognize multiple anticodons coding for a single amino acids (I.e serine 6 different anticodons and only one synthetase) • Two step process: • Activation of amino acid to aminoacyladenylate • Formation of amino-acyl-tRNA

20. Aminoacyladenylate Formation

21. Aminoacyl-tRNA Synthetase Rxn

23. Specificity of Aminoacyl-tRNA Synthetases • Anticodon and structure features of acceptor arm of specific tRNAs are important in enzyme recognition • Synthetases are highly specific for substrates, but Ile-tRNA synthetase has 1% error rate. Sometimes incorporates Val. • Ile-tRNA has proof reading function. Has deacylase activity that "edits" and hydrolyzes misacylated aminoacyl-tRNAs