Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

Abstract

An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNA;, and rabbit liver or Escherichia coh Val-tRNA vj1. Picomole quantities of fMetVal are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMetVal-His are synthesized, corresponding to the NH2-terminal sequence of a-and 3globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the a-and P-globin mRNA species.Our laboratory has recently developed a procedure for investigation of prokaryotic gene expression in an in vitro DNA-directed system by measuring the synthesis of the first di-or tripeptide characteristic of the gene product (1-5). For example, using the recombinant plasmid pNF1337, which contains the genes for Escherichia coli ribosomal proteins L10 and L12 and the (3 subunit of RNA polymerase, we studied the expression of these three genes in a coupled in vitro system by measuring the formation of fMet-Ala, fMet-Ser, and fMet-Val, respectively (1, 2, 5). The advantage of this system was that a specific di-or tripeptide product could be measured sensitively by thinlayer chromatography (1,2) or by a rapid extraction procedure (4, 5). In addition, the system only contained five purified protein factors and also could be used to quantitate the amount of active mRNA present (3). Finally, because di-or tripeptide formation indicated that proper initiation of both transcription and translation occurred, the system was ideally suited for studies on the regulation of gene expression.Crystal et aL (6)(7)(8) initially attempted to measure in vitro translation of globin mRNA in eukaryotic systems by assaying for di-and tripeptide synthesis. They did demonstrate that short NH2-terminal peptides were formed in vitro but the mRNA-dependent activity was at the borderline of detection (approximately 50 fmol) and the assay was cumbersome. With the goal of obtaining a simplified in vitro system to study eukaryotic gene expression, we have developed a highly active system for the initiation of globin mRNA translation based on the formation of the first dipeptide of the gene product. In addition, by modifying the system to obtain tripeptide synthesis, it is possible to distinguish between the expression of the a-and ,Bglobin mRNA species. Methods. All procedures were carried out at 40C unless otherwise specified.Preparation of A. salina ribosomes. Unwashed 80S ribosomes were prepared from A. salina dormant embryos according to Zasloff and Ochoa (9). The ribosomal pellets were rinsed with 50 mM Tris HCI, pH 7.4/5 mM KCI/10 mM Mg acetate/ 1 mM dithiothreitol (10), resuspended in 50 mM Tris-HCI,...

Two initiation sites detected in the small s1 species of reovirus mRNA by dipeptide synthesis in vitro.

“…The preparation of A. salina ribosomes, rabbit reticulocyte initiation factors, eukaryotic elongation factors 1 and 2 (EF-1 and -2), and the acylated tRNA species are described or referred to in a previous report (14). In the experiments reported in Table 3 and Fig.…”

“…The preparation of the various components used in the in vitro di-or tripeptide system and the characteristics of the system have been presented in detail elsewhere with globin mRNA as template (14). Each incubation mixture (60 ,ul) contained 17 mM HepesKOH at pH 7.5, 125 mM KOAc, 2.7 mM Mg(OAc)2, 1 mM dithiothreitol, 0.5 mM GTP, 1 mM ATP, 17 mM creatine phosphate, creatine kinase at 0.4 mg/ml, 2.5% polyethylene glycol 6000, 10 pmol of unlabeled fMet-tRNA1et, 10 pmol of 3H-labeled aminoacylated tRNA (3000-7000 cpm/pmol), 0.4 A260 unit of 80S ribosomes, 1 ,ug of eIF-4A, 0.27 ,ug of EF-1, various amounts of reovirus mRNA as indicated, and either 20-35 ,ug of eIFs purified by heparin-Sepharose (14) or phosphocellulose fractions A (20 ,ug), B (2 ,ug), and C (1 ,ug). For tripeptide synthesis (14), 0.15 ,ug of EF-2 and the second (unlabeled) and third (3H-labeled) aminoacyl-tRNAs were added also.…”

“…In the experiments reported in Table 3 and Fig. 3, the eukaryotic initiation factors (eIFs) isolated from the heparin-Sepharose column (14) were further fractionated by phosphocellulose chromatography as follows. The eIFs eluted from the heparin-Sepharose column were precipitated with (NH4)2SO4 (70% saturation), and the precipitate was dissolved in 20 18 hr, 700 V).…”

“…The (12). In addition, although the ribosome binding site in the 12 mRNA species has not been characterized (25), the putative initiation region at the 5' end has been reported to contain two closely spaced A-U-G-G-C-G sequences ( Dipeptide synthesis (14) was used in these studies to ascertain the initiation site of the various reovirus mRNAs in vitro. Although eukaryotic initiation does not require that the initiator Met-tRNA be formylated, in these studies we used fMet-tRNA (which can be recognized by eIFs) because fMet dipeptides can be extracted readily with ethyl acetate.…”

“…Direct demonstration of reovirus initiation sites by correlation of mRNA and polypeptide sequences has been difficult because most of the proteins in reovirions contain a blocked NH2 terminus (13). As an alternative approach, we used a modified eukaryotic translation system for measuring mRNA initiation sites based on the synthesis of the first dipeptide of the protein (14). By this procedure, it is possible to map any individual messenger-specific initiation sequence in a mixture of eukaryotic mRNAs, provided the second amino acids of the gene product are different.…”