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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