Protein Synthesis

How was experimental biochemistry used to decipher the Genetic Code?

• tRNA is an adaptor molecule that connects triplet codons in mRNA with a specific amino acid.

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

• The synthesis by Khorana 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.

How do aminoacyl-tRNA synthetases covalently link amino acids to tRNA?

• tRNA molecules are 70–90 nt in length and contain many invariant and semiconserved residues. All tRNA molecules contain an invariant CCA sequence at the 3′ end of the acceptor stem, which is the site of amino acid attachment.

• 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 are grouped into two classes depending on their structural differences, which determine whether they add the amino acid to the 2′-hydroxyl (class I) or 3′-hydroxyl (class II) of the adenosine at the terminus of the tRNA acceptor stem.

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

How do ribosomes generate nascent polypeptides from mRNA and charged tRNAs?

• 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.

• During initiation, several initiation factors are involved in assembling the ribosome and bringing the initiator methionyl-tRNA (Met-tRNAᵢ) to the P site on the ribosome.

• Elongation factors bring charged tRNAs to the A site on the ribosome (EF-Tu) and catalyze translocation (EF-G) in GTP-dependent processes. Peptide bond formation is catalyzed by rRNA.

• RF1 and RF2 are protein release factors that bind to termination codons in the mRNA to mediate translational termination; RF3 stimulates release of RF1 and RF2 and disassembly of the ribosomal complex.

• Many antibiotics inhibit bacterial translation without having a significant effect on the process in the host organism because of differences in the structures of prokaryotic and eukaryotic ribosomes.

How are proteins modified and transported throughou the cell after synthesis?

• 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 adding the modification or removing it is catalyzed by separate enzymes.

• Nuclear import and export utilize Ran proteins, importins, and exportins. Importins recognize nuclear localization signal sequences on the proteins that will be imported. Ran proteins cycle through GTP- and GDP-bound states, which alter their affinity for other proteins in the transport complexes.

• Protein translocation to the ER occurs during protein 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 posttranslationally 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 occurs in both the ER and the cytosol. N-linked glycosylation often occurs co-translationally.

• Azide-containing carbohydrate modifications can be used to identify cellular proteins. This process is known as bioorthogonal labeling and is based on click chemistry.

Everyday example: CO-25: Novel Antibiotic Discovered in Worms