PROTEIN SYNTHESIS
I. Overview of Translation
A. Translation is the process by which a polypeptide chain is synthesized by ribosomes using the sequence of codons in an mRNA to direct the sequence of amino acids.B. Translation is the ultimate step in gene expression.
C. The energy cost for protein synthesis is very high.
1. Only a small fraction of the energy input of translation is needed to form the peptide bond.
2. The majority of energy is invested to assure that the sequence of the polypeptide
is correct.
3. If incorrect polypeptides (e.g. enzymes) are made by the cell, it could have
devastating effects on cell function.
D. The mRNA is always read from 5' to 3'.
E. The polypeptide is always synthesized in the direction of amino-terminus to carboxyl-terminus.
II. Components of Translation
A. mRNA
The mRNA serves at the template that will determine the sequence of amino acids in the new polypeptide.1. Structure of mRNA
(a) 5' untranslated region (5' UTR).
(i) This is the sequence of the mRNA extending from the 5' end of the mRNA
to the initiation codon.
(ii) It is not translated into polypeptide sequence.
(iii) It has a function analogous to the function of a promoter on a gene. It will
direct the binding of the ribosome to the initiation codon.
(b) Initiation codon
(i) This is the triplet codon at which polypeptide synthesis begins.
(ii) It is always AUG and codes for a methionine.
(iii) As a result, all polypeptides are synthsized with an amino-terminal
methionine.
(c) Coding region
(i) This is the sequence of mRNA that contains the consecutive triplet codons
that direct polypeptide synthesis.
(ii) This region spans from the start codon to the stop codon.
(iii) The coding region is often referred to as the open reading frame or ORF.
(d) Stop codon
(i) This is the triplet codon that signals the termination of translation.
(ii) There are three possible stop codon sequences: UAA, UAG, UGA.
(iii) Stop codons have no corresponding tRNA or amino acid.
(e) 3' untranslated region (3' UTR)
(i) This is the nucleotide sequence downstream from the stop codon.
(ii) It extends from the stop codon to the 3' end of the mRNA
(iii) It does not code for amino acid sequence.
(iv) It may function in stabilizing the mRNA.
B. tRNA
1. The function of tRNA is deliver the correct amino acid to the ribosome as directed by the codon on the mRNA for incorporation into the polypeptide.
2. The tRNA has two important functional features
(a) A tRNA is covalently bound to an amino acid at its 3' end.
(i) This form of tRNA is called an amino acyl tRNA.
(ii) Each tRNA will be bound to only one of the twenty possible amino acids.
However, there can be multiple tRNAs for each amino acid.
(b) The tRNA contains a triplet sequence of nucleotides that are complementary to the triplet codons of the mRNA. This sequence is called the anti-codon.
(i) The covalently bound amino acid and the anti-codon strictly correspond to
one another.
(ii) As a result, the pairing of codon of the mRNA and the anti-codon of the
amino acyl-tRNA during translation will deliver the correct amino acid to
the growing polypeptide chain.
3. Structure of tRNA
(a) Cloverleaf structure
(i) 73-93 bases in length
(ii) Many unusual bases (Psuedo Uracil, Thymine, Dihdroxy Uracil etc.)
(iii) The nucleotides form many intra-chain base pairs, resulting in a secondary structure that resembles a cloverleaf.
(b) Acceptor Stem
(i) This is a region of the tRNA that is the site of attachment for the appropriate
amino acid.
(ii) It is formed by seven regular Watson & Crick base pairs between the 5' and 3' ends of the tRNA.
(iii) The 3' terminal end of all tRNAs is always CCA-OH.
(a) It is not base paired and is the site of attachment of the amino acid.
(b) The amino acid is covalently bound through an ester linkage between the carboxyl group of the amino acid and the 3' hydroxyl group of the ribose of the tRNA.
(c) Anticodon Loop
(i) The anti-codon loop contains the three nucleotide sequence that is the reverse
complement of the codon of mRNA to which it corresponds.
(ii) It consists of a total of seven unpaired bases; three of which are the anti-
codon
Wobble Hypothesis
(a) Four common bases in mRNA can be arranged in 43 or 64 different combinations(b) How does this relate to the 20 aa’s available for translation?
(i) Genetic code is redundant
(a) 3 codons used as stops UAA, UAG, UGA.
(b) 61 available as codons
(c) 1965, Francis Crick proposed the Wobble Hypothesis to account for this
inconsistency
(i) Codons in mRNA and anti-codons interact in an anti-parallel manner at the
ribosome so that positions 1 and 2 of the codon form normal Watson-Crick base pairs.
(ii) The third position allows non-standard base pairing or wobble.
(d) Consequences: Different anti-codons can pair with one codon.
Ribosome
1. Performs 3 essential functions
(a) Selection of mRNA initiation site.(b) Ensures correct pairing of codons and anticodons.
(c) Catalyzes formation of peptide bonds.
2. Ribosomes are large protein-RNA complexes that are divided into a large and small subunit.
(a) Large subunit.(i) In prokaryotes this subunit is called the 50S subunit.
(ii) In eukaryotes this subunit is called the 60S subunit.
(iii) It contains the active site for catalyzing polypeptide bond formation. This enzyme within the ribosome is called the peptidyl transferase.
(b) Small subunit
(i) In prokaryotes this subunit is called the 30S subunit.
(ii) In eukaryotes this subunit is called the 40S subunit.
(iii) It is primarily responsible for positioning the ribosome at the intitiation
codon and pairing codon and anti-codon during synthesis.
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