Initiator-elongator discrimination in vertebrate tRNAs for protein synthesis

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 TpsiC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TpsiC 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 TpsiC 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.

FIG. 1

Cloverleaf structure of the vertebrate initiator tRNA. The unique features of eukaryotic initiator tRNAs are boxed. Arrows indicate the mutations introduced. The posttranscriptional base modification at the sites of mutations are not indicated in this figure. U54 is modified to T, and U55 is modified to Ψ. In addition, U31:U39 is modified to Ψ31:Ψ39.

FIG. 2

Activities of mutant [³⁵S]Met-tRNAs in elongation in a reticulocyte cell-free system programmed with globin mRNAs. The elongation activities of the wild-type (Wt) initiator tRNA and elongator methionine are also shown. The percentage of transfer of [³⁵S]methionine to proteins is a measure of elongation activity (see text).

FIG. 3

Activities of [³⁵S]Met-tRNAs carrying G1:C72 and/or the U50C54:G63A64 mutation in the tRNA. The elongation activities of the wild-type (Wt) initiator tRNA and elongator methionine are also shown. The percentage of transfer of [³⁵S]methionine to proteins is a measure of elongation activity (see text).

FIG. 4

Cloverleaf structure of vertebrate initiator tRNA. The mutant tRNA anticodon sequences and the reporter genes designed to measure activity of these tRNAs in elongation in vivo are shown. Mutations in the tRNA acceptor and T stems are also indicated.

FIG. 5

Plasmids used for cotransfection of COS1 cells. pSVBpUC, pRSVCAT, and pCDNA1 were used for the expression of the tRNA gene, the CAT reporter gene, and the E. coli glnRS and metRS genes, respectively. The double arrows indicate the sites of insertion of the tRNA genes into the BglII site in pSVBpUC. RSV, Rous sarcoma virus.

FIG. 6

Thin-layer chromatographic analysis of CAT activities in extracts (30 μg) of COS1 cells cotransfected with SV40-based recombinant plasmids carrying the indicated initiator tRNA genes, pRSVCATM173R, and pCDMetRS. Assays from duplicate transfections are shown. The positions of acetyl-chloramphenicol (Ac-CAM) and unreacted chloramphenicol (CAM) are indicated.

FIG. 7

RNA blot hybridization of tRNAs isolated from COS1 cells cotransfected with SV40-based plasmids carrying the indicated initiator tRNA genes and either pCDNA1 (lanes 1 to 4) or pCDGlnRS (lanes 5 to 8). The tRNA and aminoacyl-tRNA species were separated on an acid urea-polyacrylamide gel.