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 2o

structure• Acceptor arm – position

where amino acid attached

• Anticodon – complementary to mRNA

• Several covalently modified bases

• Gray bases are conserved between tRNAs

tRNAs: 2o vs 3o 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 aminoacyladenylate2) Formation of amino-acyl-tRNA

Aminoacyladenylate Formation

O

N

NN

N

NH2

O

OH OH

H H

HH

O P

O-

O

OP

O-

O

O-P

O-

O

NH2

CH

C

H

O

O

PPiO-

N

NN

N

NH2

O

OH OH

H H

HH

O P

O

O

NH2

CH

C

H

O

Aminoacyl-tRNA Synthetase Rxn

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

H

N

NN

N

NH2

O

OHO

HH

HH

O

5' tRNA

NH3+

CH

C

H

O

O-

N

N N

N

NH2

O

OH OH

H H

H H

O P

O

O

NH3+

CH

C

H

O

AMP

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