Chapter 22 (Part 1) Protein Synthesis

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 substitutions in E. coli tryptophan synthase

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?

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)

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?

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

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)

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

tRNAs: 2 o vs 3 o Structure

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"

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

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

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: 1)Activation of amino acid to aminoacyladenylate 2)Formation of amino-acyl-tRNA

Aminoacyladenylate Formation

Aminoacyl-tRNA Synthetase Rxn

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