Protein Synthesis

How was the Genetic Code deciphered?

● Nirenberg and Matthaei developed in vitro translation experiments to decipher the first three codons, UUU, AAA, and CCC.

● Khorana’s synthesis of DNA molecules of known sequence, along with filter binding assays developed by Nirenberg and Leder, allowed most of the remaining codons to be decrypted.

● The standard genetic code has 64 triplet codons, 61 of which correspond with one or more of the 20 amino acids plus 3 codons that specify termination.

● Only tryptophan and methionine are encoded by a single codon each. The other 18 amino acids use from two to six codons.

● Noncanonical base pairings can occur between mRNA and tRNA at the wobble position, which is the first (5’) position of the tRNA anticodon and the third (3’) position of the mRNA codon. These alternative base pairings mean that more than one codon may be recognized by a single tRNA, in which case 61 different tRNAs are not needed in a cell.

● Inosine is produced by the deamination of adenosine and is a common modification in tRNA at the anticodon wobble position. The inosine base can form noncanonical base pairs with adenine, cytosine, and uracil.

What is the biochemistry of protein synthesis and ribozyme-mediated peptide bond formation?

● tRNAs are charged with amino acids by aminoacyl-tRNA synthetases. These enzymes catalyze a two-stage reaction in which the amino acid is first adenylated using ATP, and then the aminoacyl-adenylate is used to add the amino acid to the tRNA.

● Aminoacyl-tRNA synthetases have two types of proofreading mechanisms: one that selects for structurally similar amino acids in the active site, and one that hydrolyzes incorrect amino acids in the editing site.

● The ribosome is the site of protein synthesis in prokaryotes and eukaryotes. It is composed of two subunits that contain both protein and RNA components.

● Within the ribosome, there are three tRNA binding sites and an mRNA binding site. During elongation, a charged tRNA binds first to the A site and translocates to the P site after peptide bond formation. After transfer of the polypeptide chain, the tRNA is released from the E site.

● Prokaryotic mRNA transcripts contain a Shine–Dalgarno sequence that binds to the ribosome and positions the AUG codon in the P site. Eukaryotes scan the mRNA in a 5’ to 3’ direction until the first AUG codon is encountered, which may be within a sequence known as the Kozak sequence.

What biochemical processes are required for post-translational modification of proteins?

● Posttranslational modification can alter the structure or function of the target protein. Common modifications include phosphorylation, methylation, acetylation, glycosylation, and ubiquitination, as well as lipid modifications.

● Posttranslational modifications are often reversible, but the modification or removal of the modification is catalyzed by separate enzymes.

● Protein translocation to the ER often occurs during synthesis. The signal recognition particle (SRP) binds to a signal sequence at the N terminus of the nascent polypeptide and directs the ribosome to the SRP receptor in the ER membrane. As the remainder of the protein is synthesized, it directly enters the ER lumen.

● Proteins that must attach to a membrane as part of their function are post-translationally modified in the ER by the attachment of one or more lipid residues, such as farnesyl, geranylgeranyl, palmitoyl, or myristoyl residues.

● N-linked and O-linked glycosylation can occur both in the ER and the cytosol. N-linked glycosylation often occurs co-translationally.

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