Elongation factor Ts (EF-Ts) is the guanine nucleotide exchange factor for elongation factor Tu (EF-Tu). An important feature of the nucleotide exchange is the structural rearrangement of EF-Tu in the EF-Tu⅐EF-Ts complex caused by insertion of Phe-81 of EF-Ts between His-84 and His-118 of EF-Tu. In this study, the contribution of His-118 to nucleotide release was studied by pre-steady state kinetic analysis of nucleotide exchange in EF-Tu mutants in which His-118 was replaced by Ala or Glu. Intrinsic as well as EF-Ts-catalyzed release of GDP/ GTP was affected by the mutations, resulting in an ϳ10-fold faster spontaneous nucleotide release and a 10 -50-fold slower EF-Ts-catalyzed nucleotide release. The effects are attributed to the interference of the mutations with the EF-Ts-induced movements of the P-loop of EF-Tu and changes at the domain 1/3 interface, leading to the release of the ␤-phosphate group of GTP/GDP. The K d for GTP is increased by more than 40 times when His-118 is replaced with Glu, which may explain the inhibition by His-118 mutations of aminoacyl-tRNA binding to EF-Tu. The mutations had no effect on EF-Tu-dependent delivery of aminoacyl-tRNA to the ribosome.

BackgroundThe oncogene PTI-1 was originally isolated from a prostate cancer cell line by its capability to transform rat fibroblasts. The PTI-1 mRNA has a very eccentric structure as the 5′UTR is similar to prokaryotic 23S rRNA, while the major open reading frame and the 3′UTR corresponds to a part of the mRNA encoding human translation elongation factor eEF1A1. Thus, the largest open reading frame encodes a truncated version of eEF1A1 lacking the first 67 amino acids, while having three unique N-terminal amino acids. Previously, the UTRs were shown to be a prerequisite for the transforming capacity of the PTI-1 transcript. In this study, we have investigated the possible role of the UTRs in regulating protein expression and localization.MethodsThe protein expression profiles of a number of PTI-1 mRNA variants were studied in vitro and in vivo. Furthermore, the oncogenic potentials of the same PTI-1 mRNAs were determined by monitoring the capacities of stably transfected cells expressing these mRNAs to induce tumors in nude mice and form foci in cell culture. Finally, the cellular localizations of PTI-1 proteins expressed from these mRNAs were determined by fluorescence microscopy.ResultsThe PTI-1 mRNA was found to give rise to multiple protein products that potentially originate from translation initiation at downstream, inframe AUGs within the major open reading frame. At least one of the truncated protein variants was also found to be oncogenic. However, the UTRs did not appear to influence the amount and identities of these truncated protein products. In contrast, our localization studies showed that the UTRs of the transcript promote a nuclear localization of the encoded protein(s).ConclusionsTranslation of the PTI-1 mRNA results in multiple protein products of which (a) truncated variant(s) may play a predominant role during cellular transformation. The PTI-1 UTRs did not seem to play a role in translation regulation, but appeared to contribute to a nuclear localization of the PTI-1 protein(s). This indicates that the PTI-1 protein(s) exert(s) its/their oncogenic function inside the nucleus.