Molecular Biology

Chapter 18 Outline

tRNA binding sites in the ribosome:

∑ Puromycin is an amino acid coupled to an adenosine analog; when used to charge a tRNA, it can prematurely terminate translation; Puromycin can not bind to the ribosome until the peptidyl tRNA has translocated to the P site after peptide bond formation ≠ showing that there are two sites for tRNA binding (F18.12); A third exit (E) tRNA binding site was identified by showing that 3 molecules of tRNA^Phe could bind to a ribosome translating a poly-U mRNA (T18.2).

Binding an Aminoacyl tRNA to the A site:

∑ EF-Tu and EF-Ts are required for binding aminoacyl-tRNAs to the ribosome; EF-Tu/Ts requires GTP to bind to the ribosome (F18.14a); Hydrolysis of GTP by EF-Tu/Ts is not required for binding aminoacyl tRNA to the ribosome, but is required for peptide bond formation (T18.3); EF-Tu/Ts-GTP complexes will bind to a nitrocellulose filter, but adding Phe-tRNA will release them from the filter ≠ indicating a EF-Tu/Ts-GTP-Phe-tRNA^Phe ternary complex (F18.16); The ternary complex would only form with charged tRNAs destined for binding to the A site (F18.17); EF-Ts is a GEF that exchanges GTP for GDP on EF-Tu (F18.19); Model for binding tRNAs to the A site (F18.15).

∑ Part of the accuracy of protein synthesis comes from proofreading at the ternary complex binding stage; Proofreading initially occurs at the binding step, described by the k_-1 binding constant (F18.21); Proofreading also occurs after binding by releasing an aminoacyl-tRNA from the A site before peptide bond formation has occured, described by k₄ (F18.21); Speed and accuracy are inversely related; The error rate for translation in E. coli is 0.01%.

Peptide bond formation:

∑ The peptidyl transferase that catalyzes the peptide bond is part of the ribsosome; Puromycin will be added to a growing polypeptide chain via the action of peptidyl transferase (F18.22a); After isolating large ribosomal subunit by incubation in low Mg⁺², adding puromycin results in peptide bond formation ≠ showing the 50S subunit contains peptidyl transferase (F18.22b); most proteins in the large ribosomal subunit are dispensible for peptidyl transferase activity (F18.24), but is sensitive to peptidyl transferase inhibitors and RNase (F18.25) ≠ indicating that peptidyl transferase is catalyzed by the 23S rRNA itself; 23S rRNA, L2 and L3 proteins are sufficient to reconstitute peptidyl transferase activity.

Translocation:

∑ EF-G is required for translocating the mRNA after peptide bond formation (F18.14b); Translocation moves the mRNA three nucleotides based on which parts of the mRNA are protected by the ribosome before and after translocation (F18.26); GTP hydrolysis is required for translocation to occur (F18.27 ≠ model I).

Termination:

∑ Termination occurs at stop codons ≠ UAG, UAA and UGA; Stop codons can be suppressed by tRNAs mutated to have anticodons complementary to stop codons (F18.35); Stop codons are recognized by release factors; Release factors were found by incubating ribosomes containing a labeled peptide, whose next codon was a stop codon, with ribosomal supernatant fractions (F18.36); There are two release factors (RF-1 and RF-2) that recognize the stop codons with different specificities (T18.6); Another release factor (RF-3) is a ribosome-dependent GTPase that helps RF-1 and RF-2 bind the ribosome; RF-1 and RF-2 mimic tRNAs by recognizing stop codons using a tripeptide determinant (F18.38); Eukaryotic release factors include eRF1, which recognizes all of the stop codons, and and eFR-3, which is the ribosome-dependent GTPase.

GTPases and translation:

∑ eIF2, EF-Tu and RF3 are all GTPases that allow either aminoacyl-tRNAs or release factors to bind to anticodons in the ribosome; These proteins all belong to a large class of G-proteins that (1) bind GDP/GTP, (2) have different conformation when bound to GTP, GDP or unbound, (3) They carry out their function when bound to GTP, (4) They are GTPases, (5) Their GTPase activity is stimulated by another factor, (6) When they hydrolyze their GTP they become inactivated, and (7) they are reactivated by GEFs (F18.30).