Principal Investigator Uttam RajBhandary
We showed that mutant initiator tRNAs with changes in the anticodon sequence can initiate protein synthesis from an initiation codon that is complementary to the tRNA anticodon and with amino acids other than methionine. This finding made possible an assay for activity of mutant initiator tRNAs in vivo. We have identified the critical elements in the tRNA necessary for specifying all of its distinctive properties including the role of the unique features common to all eubacterial initiator tRNAs. In identity swap experiments, we have shown that introduction of these unique features into E. coli elongator methionine tRNA converts it into a fully active initiator tRNA in vivo.
In the process of this work, we have generated mutant initiator tRNAs which are blocked at each of the steps in the initiation pathway. Using these mutants, we have identified intragenic and extragenic suppressors which can rescue the initiation defects of some of the mutant tRNAs and have identified the important role of the amino acid methionine attached to the tRNA in recognition of the tRNA by the formylating enzyme and by the initiation factor IF2. We are now interested in structural analysis of the initiator tRNAs in a complex with various proteins such as the initiation factor IF2.
Other studies underway include an analysis of the requirements in initiator tRNA and in initiation factors for “de novo initiation”, which involves the assembly of mRNA, ribosome and initiator tRNA, versus “reinitiation”, in which the ribosome that has finished translation of an open reading frame and is bound to the mRNA, continues to translate the next open reading frame. We have identified some of the requirements in initiator tRNA and initiation factors for reinitiation.