Translational Regulation of Gene Expression
- Initiation
- Elongation
- Termination
Translational Assembly of Amino Acids into Polypeptides
-amino acids contain an amino and carboxyl group bonded to a central carbon (alpha) with a hydrogen and a R group
-R group is variable and determines unique character of amino acid
-two amino acids are joined together by a covalent “peptide” bond between the amino and carboxyl by a dehydration reaction
-polypeptides are linear chains of amino acids linked by peptide bonds
Non-polar Amino Acids
-R groups usually containing -CH2 or -CH3
Uncharged Polar Amino Acids
-R groups usually contain oxygen (or -OH)
Charged Amino Acids
-R groups that contain acids or bases that can ionize
Aromatic Amino Acids
-R groups containing a carbon ring with alternating single and double bonds
Special Functional Amino Acids
-Include:
- Methionine: first amino acid added during translation
- Proline: causes kinks (bends) in polypeptide
- Cysteine: 2 cysteines can form covalent bonds between the S atoms to make a disulfide bridge
Levels of Protein Structure
-primary amino acid sequence determines protein folding and 3-D structure which is critical for proper function
-secondary structure depends on hydrogen bonding in the polypeptide backbone (alpha-helices and beta-sheets) - sequence independent
-tertiary structure is the 3-D structure of a single polypeptide and is composed of interactions between amino acid side chains
-quaternary structure are interactions between more than one polypeptide to form a multi-subunit protein
-protein folding is disrupted by denaturation (heat and chemicals) or mutations that change amino acid sequence
tRNAs
-adaptors between codons (mRNA) and amino acids (non-coding)
-2-D cloverleaf and 3-D L-shaped folded RNA molecule from self complementarity
tRNA: Acceptor Stem
-where the amino acid is attached and contains the sequence 5’-CCA-3’ at the 3’ end of tRNA
tRNA: Anticodon
-the bottom loop of the cloverleaf and is a three-nucleotide sequence that base pairs to the codon on the mRNA
Aminoacyl-tRNA Charging
-Aminoacyl-tRNA synthetase adds an amino acid to the acceptor stem of the correct tRNA
-there is a unique aminoacyl-tRNA synthetase for each of the 20 amino acids
-charging reaction (aminoacylation):
Amino acid + tRNA + ATP —> aminoacyl-tRNA + AMP + PPi
The Genetic Code
-consists of codons and the amino acids specified by these codons
-the codons are written 5’ to 3’, as they appear in the mRNA
-61 sence codons
-3 termination/stop/nonsense codons that do not code for amino acids (transfer RNA does not bind to these codons)
-genetic code shows degeneracy in which an amino acid can be specified by more than one codon
Rules of the Genetic Code
- codons on the mRNA are read in the 5’ to 3’ direction
- codons are non overlapping and the message contains no gaps ‘
- message is translated in a fixed reading frame set by the start codon
- amino acids added amino to carboxyl
Wobble in the tRNA Anticodon
-some tRNAs can read more than 1 codon
-the base at the 5’ end of the anticodon can H-bond with more than one type of base at the 3’ end of a codon : wobble
-pairing of the other two nucleotides in the anticodon with the codon is precise
Pair Combinations With Wobbling
G - U or C
C - G
A - U
U - A or G
Inosine - A, U, or C
Ribosome Protein Synthesis Machinery
-composed of 2 subunits made of a complex of rRNA and protein
-large subunit (50S: prokaryotes; 60S eukaryotes): made of rRNA + proteins and contains the peptidyl-transferase centre for formation of peptide bonds
-small subunit (30S: prokaryotes; 40S: eukaryotes): made of rRNA + proteins and contains decoding centre where charged tRNAs read the codon of the mRNA
-S (svedberg unit): measure of sedimentation velocity and therefore, mass (large things sediment faster)
-each subunit exists separately in the cytoplasm, but join on the mRNA molecule
tRNA Binding Sites of Ribosomes
- A site (aminoacyl): binds to the tRNA carrying the next amino acid
- P site (peptidyl): binds to the tRNA attached to the growing polypeptide
- E site (exit): binds the tRNA that carried the previous amino acid
Translation Initiation (Eukaryotes)
- indicator tRNA (met) and GTP are brought to the P-site of the small ribosomal subunit
- complex of initiator tRNA (met) and small ribosomal subunit is recruited to the capped 5’ end of mRNA and scans along the mRNA in a 5’-3’ direction until it reaches the start codon
- complementary base pairing occurs between the anticodon of initiator tRNA (met) and the start codon on the mRNA
- large ribosomal subunit binds to the small subunit to form the initiation complex, which is now ready to accept the next tRNA in the A site
- GTP is hydrolyzed and translation begins
Translation: Elongation
- Elongation Factor (EF-GTP): adds a charged aminoacyl tRNA to the A-site with the hydrolysis of the GTP
-the anticodon of the incoming tRNA must be complementary to the codon on the mRNA
- The Peptidyl Transferase Centre of the large subunit forms a peptide bond between the carboxyl group of the amino acid in the P-site and the amino group of the amino acid in the A-site
-the tRNA site is now uncharged (empty)
- GTP hydrolysis translocates (shifts) the ribosome three nucleotides along the mRNA (5’ to 3’ direction)
-the empty tRNA is now in the E-site and is ejected
-the tRNA with the growing polypeptide is now in the P-site
-the A-site is now vacant
- the next codon of the mRNA is now aligned with the A-site and the next aminoacyl tRNA can be loaded by EF-GTP
Translation: Termination
- one of the three termination (stop) codons are found at the 3’ end of the mRNA
-UAA, UAG, and UGA
- There are no tRNAs for the stop codons
- When the ribosome reaches the stop codon, release factor (RF) enters the A-site
- RF stimulates peptidyl transferase to cleave the polypeptide from the P-site tRNA
-the polypeptide is released from the ribosome
- the translational machinery dissociate from the mRNA
-termination releases a completed polypeptide from the ribosome
Post-Translational Regulation of Gene Expression
- Phosphorylation
- Ubiquitination
- Proteolysis
Phosphorylation
-addition of phosphate to proteins by kinase can activate or inhibit
-phosphatases remove phosphate
-pyruvate kinase catalyses the last reaction of glycolysis
-phosphorylation of pyruvate kinase turns the enzyme “off”
-dephosphorylation of pyruvate kinase turns the enzyme back “on”
Ubiquitination
-addition of ubiquitin molecules to proteins will target them for destruction by proteosomes (turn proteins to amino acids)
-ubiquitin is a small protein that can be covalently attached to other proteins
