Protein Synthesis -- Translation
I.
CHEMISTRY
A. Amine groups of amino acids added to carboxyl group of the peptide chain
1. Dehydration synthesis reaction
2. Peptide bond formation
3. Catalyzed by ribosomes
II.AMINO
ACID SEQUENCE
A. General
1. The amino acid sequence of every protein is encoded for by the nucleotide
sequence of DNA
2. DNA’s nucleotide sequence is not used directly, but is transcribed into mRNA
3. There are 20 amino acids that are commonly found in proteins
B. The Genetic Code
1. Definition
a) Nucleotide sequences (codons) correspond to specific amino acid types and
translational signals
2. The codon
a) There are 20 amino acids and 4 nucleotide bases
(1) Obviously single nucleotides cannot code for amino acids
(2) There is 16 combinations possible for 4 nucleotides read 2 at a time, not
enough to code for 20 amino acids
(3) There are 64 combinations possible for 4 nucleotides read 3 at a time, enough
to code for 20 amino acids and translational signals
(4) There are 256 combination possible for 4 nucleotides read 4 at a time, way
more than enough, and wasteful of genome size
b) Codons are sequences of three nucleotides
(1) Three bases are read sequentially to code for an amino acid
c) Redundancy
(1) There are 64 possible codons and only 20 amino acids and all possible
codons are found in RNA
(2) Several different nucleotide sequences (codons) can code for the same amino
acid
(a) This is known as genetic code redundancy
(b) Codons that code for the same amino acid usually differ in just the last
nucleotide
(i) Leucine codons differ in last and second to last nucleotides
(ii) Methionine and tryptophan each have only one codon
(3) Silent mutations
(a) A mutation which changes a codon, but so that it still codes for the same
amino acid, is referred to as a silent mutation
(i) Genotype is changed, phenotype stays the same
d) Special codons
(1) Stop codons
(a) Three codons do not code for any amino acid
(b) UAA, UAG, UGA
(c) These are the site that ribosomes stall on mRNA molecule, resulting in
termination of translation
(2) Start codons
(a) AUG is referred to as a start codon
(i) This is where translation begins
(ii) AUG codes for methionine
(b) Each protein starts with a f-met amino acid at its N-terminus
(i) This is often cleaved during processing
e) Reading frame
(1) Since codons are read sequentially, an addition or deletion of just one
nucleotide would result in a “shift in reading frame”
(a) Example
UUUCCCAAAGGG codes for Phe-Pro-Lys-Gly
AUUUCCCAAAGGG codes for Ile-Ser-Gln-Arg
UUCCCAAAGGG codes for Phe-Pro-Lys
(2) Hence, a single base pair addition of deletion in DNA would result in a protein
with a completely different amino acid sequence
(a) Such a protein would be non-functional
(b) This is referred to as a missense mutation
(3) Frame shift mutations can often change a codon from coding for an amino
acid to a stop sequence
(a) Such a protein would be shorter than the normal protein
(b) This is referred to as a nonsense mutation
f) Overlapping genes
(1) Some viruses use the same DNA sequence to code for two different proteins
(a) These proteins may have different amino acid sequences if read in
different frames
3. Determination of genetic code
a) Accomplished in vitro with synthetic RNA
(1) Poly-U resulted in polyphenylalanine
(a) Hence, UUU is the codon for phenylalanine
(2) Poly-U with 3’ G-terminus resulted in polyphenylalanine with C-terminal
leucine
(i) Hence, UUG is codon for leucine
4. Universality of the genetic code
a) A specific codon codes for the same amino acid, regardless of the species
(1) The genetic code is the same for viruses, bacteria, protists, fungi, plants and
animals
(2) The genetic code is thought of as almost universal
b) Exceptions
(1) There are a few species specific exceptions in some mitochondria and a few
yeast and ciliates
III.TRANSFER
RNA AS ADAPTER MOLECULES
A. Amino acids do not have direct affinity for nucleic acids
1. Therefore, mRNA can not directly serve as a template for protein synthesis
2. There must exist “adapter molecules” which can read the RNA sequence
(codons) and bring with it the correct amino acids
a) This is the function of tRNA molecules
B. General structure
1. 73 to 93 nucleotides long
2. Intrastrand complimentarily causes three stem-loop structures to form (often
drawn as a clover leaf)
C. Regions of tRNA molecules
1. Anticodon
a) One loop contains three nucleotides that interact with mRNA during
translation
(1) These three nucleotides of tRNA are referred to as an anticodon
(2) The loop containing the anticodon is referred to as the anticodon loop
2. Amino acid attachment site
a) Near the 3’ terminus is a sequence CCA
b) Specific amino acids are covalently attached here by enzymes called
aminoacyl-tRNA synthetases
3. Aminoacyl-tRNA synthetase recognition regions
a) Different aminoacyl-tRNA synthetases must match a specific amino acid
with a specific tRNAs and their anticodons
(1) Therefore, there should be a region of tRNA that will bind only with the proper
aminoacyl-tRNA synthetase
D. Sixty-one different tRNAs
1. Differ in their anticodon sequence, amino acid attached, and their aminoacyltRNA synthetase recognition sequence
a) There are no tRNA molecules with anticodons for the stop codons (UAG,
UAA, UGA)
E. Aminoacyl-tRNA synthetases
1. More than twenty different enzymes
a) At least one for each amino acid
(1) Possibly more for amino acids with more than one codon
b) Aminoacyl-tRNA synthetases and tRNAs are named for the amino acid
associated with them
(1) Example, leucyl-tRNA synthetase attaches leucine to tRNALeu
2. Terminology
a) Charged tRNA
(1) One with the correct amino acid attached
(2) Also referred to as acylated
b) Uncharged tRNA
(1) One with no amino acid attached
c) Mischarged tRNA
(1) One with the incorrect amino acid attached
F. Fidelity of the genetic code
1. Requires correct amino acid attachment to tRNAs
2. Requires accurate codon-anticodon binding
G. Anticodon binding to codon
1. General
a) The anticodons of tRNAs are complimentary to the codons of mRNA
b) It is this complimentarily that is essential for tRNA binding to mRNA, not the
amino acid attached to it
c) This is demonstrated by the two following experiments
2. Mischarged tRNA
a) tRNACys molecules were converted to tRNAAla
(1) These were used in vitro during translation of hemoglobin RNA
(2) The hemoglobin consistently contained alanine where it normally contained
cysteine
b) Shows that anticodon is essential in binding to codon, not the specific
amino acid attached to the tRNA
3. Alteration of anticodon
a) The anticodon of tRNA loaded with its correct amino acid (UCC – glycine)
was altered so that the anticodon no longer matched its specific amino acid
(1) UCU – glycine instead of the correct UCU – arginine combination
b) When used in translation, the protein would contain glycine where it
normally had arginine, and would not add glycine at the proper locations
4. Wobble
a) If the first two nucleotides of the anticodon binds to the codon, the third pair
has some flexibility
b) Inosine can bind to A, U, and C
IV.RIBOSOMES
A. Composition
1. Two subunits that can dissociate
a) Svedberg unit
2. Contains many proteins and several RNA molecules
B. Prokaryotes
1. 70S ribosome
a) 50S subunit
(1) 32 proteins
(2) 23S and 5S rRNA molecules
b) 30S subunit
(1) 21 proteins
(2) 16S rRNA
C. Eukaryotes
1. 80 ribosome (more variability between species)
a) 60S subunit
(1) 50 proteins
(2) 5S, 5.8S, and 28S rRNA molecules
b) 40S subunit
(1) 30 proteins
(2) 18S rRNA
D. Enzyme
1. Form peptide bonds
E. Scaffold
1. P (peptidyl) tRNA binding site
2. A (aminoacyl) tRNA binding site
V.
PROTEIN SYNTHESIS
A. INITIATION
1. 30S subunit of ribosome binds to mRNA
2. Site of initiation
a) Prokaryotes
(1) Ribosomes bind to the Shine-Delgaro sequence upstream from AUG
(a) Sometimes referred to as the ribosomal binding sequence
b) Eukaryotes
(1) Cap binding proteins attract 40S ribosomal subunit
(2) RNA is monocistronic (though possibly a complex transcriptional unit)
3. tRNA-methionine binds to mRNA-ribosome (30S or 40S) complex
4. 50S subunit binds to complex
a) tRNA-methionine occupies the P site
B. ELONGATION
1. Amino acid-tRNA binds to A-site
a) tRNA must have proper anticodon
b) H-bonds between codon and anticodon nucleotides
2. Peptide bond formation
a) Amino acid in P-site is attached to the amino acid in A site
(1) Covalent bond
(2) Catalyzed by peptidyl transferase
3. Translocation
a) Ribosome moves down mRNA to next codon
(1) tRNA in P-site is released
(2) tRNA in A-site is moved to P-site
(a) A-site is now empty
4. Elongation steps repeat, adding more amino acid
C. TERMINATION
1. Ribosome stalls when it reaches a stop codon (UAA, UAG, or AGA)
a) Interactions with another protein, the releasing factor causes the ribosome
to dissociate, releasing the newly synthesized protein
2. Polycistronic mRNA
a) In prokaryotes, the ribosome does not completely dissociate, and it will
come together at the next start codon
b) The further the start codon is from the stop codon, the less likely translation
will be re-initiated
VI.PHYSICAL
STRUCTURES IN TRANSLATION
A. Polysomes
1. After the ribosome has move about 75 bases down the RNA from the ShineDelgaro or cap-binding proteins, these are free to bind another ribosome
2. A mRNA normally contain many ribosomes at a given time
a) The protein attached to the ribosome are longer the closer the ribosome is
to the 3' end of the mRNA
B. Coupled transcription / translation
1. Since translation starts from the 5' end of RNA, which is translated first, and
bacteria do not have a nuclear membrane, transcription and translation can
occur simultaneously
a) Before transcription of a RNA molecule is completed, it is already being
translated
2. This allows for the attenuation method of transcriptional regulation
a) An example is the histidine operon
VII.ANTIBIOTICS
A. Aminoglycosides
1. Examples
a) Streptomycin, Gentamycin, Neomycin, Kanamycin
2. Binds to 30S ribosomal subunit
a) Prevents initiation complex from forming
b) Induces misreading
B. Tetracyclines
1. Binds to 30S ribosomal subunit
a) Blocks amino acid-tRNA from entering the A site
C. Chloramphenical
1. Binds to 50S ribosomal subunit
a) Prevents peptide bond formation
b) Inhibits peptidyl transferase
D. Macrolids
1. Example
a) Erythromycin
2. Binds to 50S subunit
a) Prevents translocation