TY - JOUR
T1 - Antizyme frameshifting as a functional probe of eukaryotic translational termination
AU - Karamysheva, Zemfira N.
AU - Karamyshev, Andrey L.
AU - Ito, Koichi
AU - Yokogawa, Takashi
AU - Nishikawa, Kazuya
AU - Nakamura, Yoshikazu
AU - Matsufuji, Senya
N1 - Funding Information:
The authors thank S. Hoshino for providing human eRF3 clone, R. Mattes for pUBS520 plasmid and C. Crist for helping preparation of the manuscript. This work was supported in part by grants from the following: the Ministry of Education, Sports, Culture, Science and Technology of Japan (MEXT) to Y.N. and S.M.; the Human Frontier Science Program to Y.N.; the Basic Research for Innovation Biosciences Program of the Bio-oriented Technology Research Advancement Institution (BRAIN) to Y.N.; and the Organization for Pharmaceutical Safety and Research (OPSR) to Y.N. A.L.K. was a postdoctoral fellow of the Japan Society for the Promotion of Science and Z.N.K. was a research fellow of the Research for the Future Program of the Japan Society for the Promotion of Science.
PY - 2003/10/15
Y1 - 2003/10/15
N2 - Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.
AB - Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.
UR - http://www.scopus.com/inward/record.url?scp=0344874263&partnerID=8YFLogxK
U2 - 10.1093/nar/gkg789
DO - 10.1093/nar/gkg789
M3 - Article
C2 - 14530443
AN - SCOPUS:0344874263
SN - 0305-1048
VL - 31
SP - 5949
EP - 5956
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 20
ER -