10 - Genetic Code & Translation

AUG; it codes for methionine, and sets the reading frame for translation from the mRNA

One base is replaced by another; when there is no change in the AA produced it is called a silent mutation; if one AA is replaced by another it is called a missense mutation; when the code for one AA is replaced by a stop codon it is called a nonsense mutation

One or more bases are added to the sequence; if added in multiples of three AAs are added, when not added in multiples of three, it results in a frame shift mutation

One or more bases are deleted; effects are similar to that of insertion

Amino acids, tRNA, mRNA, rsomes, aminoacyl-tRNA synthetases, protein factors, ATP, and GTP

Carries AAs to the site of protein synthesis

Carries genetic information from genes to the cytoplasm for translation

Also known as a polysome; clusters of rsomes translating a single mRNA

The small subunit binds the mRNA and tRNAs and the large subunit catalyzes peptide bond formation

It enables the initiation at the correct start site in prokaryotes and proofreads the correct codon-anticodon base pairing; ensures that each successive codon in mRNA engages precisely with the anticodon and does not slip by a nucleotide; it catalyzes the formation of the peptide bond

There are three for tRNAs and one for mRNA

• Aminoacyl-tRNA-binding site (A-site); binds the incoming tRNA molecule linked to an AA
• Peptidyl-tRNA-binding site (P-site); binds tRNA molecule that is linked to the growing peptide chain
• “Exiting”-tRNA-binding site (E-site); binds tRNA molecule that is not “free”

• 1. AA activation, which involves the charging of a tRNA w/ its cognate AA
• 2. Initiation, which is the assembly of the
• components of the translational machinery before peptide bond formation
• 3. Elongation, which is the addition of AAs
• 4. Termination, which is the disassembly of the translational machinery and the release of the pp chain

Activation is catalyzed by specific aminoacyl-tRNA synthetases; each aminoacyl tRNA synthetase attaches a single AA to all of the cognate tRNAs; AA + tRNA + ATP → Aminoacyl-tRNA + AMP + 2Pi

N-terminus to C-terminus direction; the mRNA is read in the 5’ to 3’ direction

AUG; which codes for Met; initiator tRNA recognizes the initiation codon; this is different from the tRNA that incorporates Met (tRNAf-Met in prokaryotes and tRNAMeti in eukaryotes)

Found in prokaryotes, this is a region of mRNA that is upstream of the initiation codon, and it base pairs with the 3’ end of the 16S rRNA; it positions the start codon in the P-site of the rsome; it is found before the first AUG of each cistron in the polycistronic mRNA

The sequence is PuCCAUGG, where Pu is the purine nucleotide A or G; this is recognized by the ribosome as the translational start site; the sequence aids in defining the initial AUG codon for translation, the loss of which reduces the efficiency of translational initiation

The association of mRNA, f-Met‐tRNA^f‐Met, and the ribosomal subunits

By a series of protein factors called initiation factors (IFs); there are three prokaryotic initiation factors which are called IF-1, IF-2, and IF-3

• 1. IF-3 binds the 30S rsome subunit, preventing it from prematurely complexing with the 50S subunit; IF-1 assists in this endeavor
• 2. IF-2 complexed to GTP (IF2-GTP) binds f-Met‐tRNAf‐Met and helps it to dock with 30S
• 3. As the mRNA binds, IF-3 helps to correctly position the complex such that the f-Met‐tRNAf‐Met interacts via base pairing with AUG at the P-site
• 4. As 50S joins the complex, IF2-GTP is hydrolyzed, and 50S serves as the GTPase activating protein for IF-2
• 5. A complex consisting of the 70S rsome, mRNA, and f-Met‐tRNAf‐Met base paired with mRNA at the P-site will be formed; there will be no more Ifs or GTP or GDP bound to it

• 1. A pre-initiation complex composed of several initiation factors, small rsome subunit, initiator tRNA, and Met-tRNAMeti is formed
• 2. The pre-initiation complex binds to the mRNA at the 5’ cap, and translocates along the mRNA in the 5ʹ → 3′ direction by a process called scanning, until the initiation codon is reached; this is facilitated by eIF4A
• 3. GTP bound to eIF2 is hydrolyzed as the large ribosomal subunit joins the complex
• 4. A complex consisting of the complete 80S ribosome, mRNA, and Met-­‐tRNAMeti base paired with mRNA at the P‐site is formed

• 1. EF-Tu/eEF1A-GTP binds and delivers an
• aminoacyl-tRNA to the A-site
• 2. The interaction of EF-Tu/eEF1A-GTP-aminoacyl-tRNA with the rsome hydrolyzes the GTP on  EF-Tu/eEF1A-GTP-aminoacyl-tRNA to GDP + Pi, EF-Tu/eEF1A undergoes a large conformational change and the complex dissociates leaving the aminoacyl-tRNA at the A-site with the amino group of the AA near the 3’ end of the peptidyl tRNA
• 3. The ribozyme 23S rRNA catalyzes peptide bond formation between the amino N of the AA linked to the tRNA in the A-site to the
• carbonyl C of the AA in ester linkage to the tRNA in the P-site (this one loses
• it’s tRNA)
• 4. Once the peptide bond formation is complete, the E-site is empty, the P-site is occupied by the tRNA that unloaded the peptide, and the A-site is occupied by the tRNA which is attached to the nascent PP
• 5. EF-G/eEF2 is activated through its interaction with the rsome, and hydrolyzes its bound GTP to GDP + Pi; this causes EF-G/eEF2 to dissociate from the rsome
• 6. Step 5 cases the tRNA attached to the nascent PP to be pushed from the A-site to the P-site, and the empty tRNA that was in the P-site shifts to the E-site
• 7. The A-site is now open for the next EF-Tu/eEF1A-GTP-aminoacyl-tRNA to come and base pair with the next codon

EF-T/eEF1B functions to reactivate eEF1A; it causes eEF1A to release its bound GDP; when eEF1A dissociates from eEF1B, then eEF1A binds a fresh GTP

Protein synthesis is terminated when the translocation reaction brings a stop codon to the A site of the ribosome; this requires the release factors RF/eRF (prokaryotes/eukaryotes); they allow the nascent PP chain to be released

The release factors recognize stop codons and occupy the A site; this changes the specificity of peptidyl transferase such that the growing peptide chain is transferred from the peptidyl--‐tRNA to H2O, thus releasing the nascent peptide chain

After releasing the nascent PP and the release factors the rsome is still bound to the mRNA and two “empty” tRNAs in the E and P-sites; to begin translating again a ribosomal recycling factor (RRF in prokaryotes and eRRF in eukaryotes) is required to release the tRNAs and mRNA from the rsome and allow the rsome to separate into subunits; this separation is required to begin translating again

Viruses shut off translation of host mRNA and promote the translation of viral RNA; they code for proteins that either inhibit protein‐protein interaction among the various proteins involved in mRNA cap-dependent translation initiation or degrade the eukaryotic initiation factors

It has a protease which specifically cuts eIF4G and inhibits translation initiation; the viral mRNA is translated b/c it has an internal rsome entry site (IRES) where the rsome can bind and start translating viral mRNA

It is found in proteins destined for the ER, lysosomes, secretion, or membranes; this is a sequence in the approximately first 20 AA residues, which allows that region of the protein molecule to pass through the plasma membrane; in proteins this hydrophobic signal is retained and helps in anchoring the protein to the membrane; in secretory proteins, the N‐terminal signal sequence is cleaved and the protein is released into the lumen of the ER; glycosylation and packaging of the proteins into vesicles takes place in the Golgi

Degradation of misfolded proteins and cell cycle regulators; main mechanism for catabolism of proteins; closely tied to the functionalities of the cell cycle, gene expression, immune and/or stress response, and apoptosis

Conjugation (the process of targeting a substrate protein with several ubiquitin molecules) and degradation

ATP dependent; the first Ub is attached to the Lys residue on the target protein; addition Ub molecules are attached to the previous Ub molecule to form a chain

The ubiquinated substrate protein enters the proteasome, which is a hollow, cylindrical complex with 3-7 protease active sites on the interior; the complex binds to the polyUb proteins and regulatory proteins in the complex release the Ubs for reuse; the protein is unfolded using the energy of ATP, and is then translocated into the main chamber; the protease sites cleave the peptide at specific points producing a set of peptides which are ~8 AAs long

Several neural diseases; may lead to brain tumors such as astrocytomas

Results in the accumulation of proteins and plaque buildup, leading to neurodegenerative diseases such as Alzheimer's, Parkinson’s and Huntington’s diseases

Results in the arrest of cell cycle progression; this is being utilized to kill cancer cells