Toward an understanding of the formylation of initiator tRNA methionine in prokaryotic protein synthesis. I. In vitro studies of the 30S and 70S ribosomal-tRNA complex

Biochemistry ◽  
1976 ◽  
Vol 15 (7) ◽  
pp. 1357-1362 ◽  
Author(s):  
Hans Uffe Petersen ◽  
Antoine Danchin ◽  
Marianne Grunberg-Manago
Keyword(s):  
Protein Synthesis ◽  
In Vitro Studies ◽  
Initiator Trna ◽  
Prokaryotic Protein

scholarly journals Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

1998 ◽  
Vol 18 (3) ◽  
pp. 1459-1466 ◽  
Author(s):  
Harold J. Drabkin ◽  
Melanie Estrella ◽  
Uttam L. Rajbhandary
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Elongation Factor ◽  
Reporter System ◽  
Base Pairs ◽  
Initiator Trna ◽  
Phosphate Backbone ◽  
Elongation Step

ABSTRACT Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.

Sign in / Sign up

Export Citation Format

Share Document