Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Abstract

A simplified translation system coupled to DNA transcription that involves assaying the synthesis of the first dipeptide of a gene product has been described recently Procx NatL Acad. Sci USA 78,[4261][4262][4263][4264]. Using this dipeptide system, we have investigated the expression ofgenes carried on plasmids coding for (8-lactamase, ribosomal protein L12, and the chloroplast large subunit (LS) of ribulosebisphosphate carboxylase (RbuBPCase). Although all three nascent gene products begin with the sequence fMet-Ser, the formation of fMet-Ser can be used to distinguish between the synthesis of .8-lactamase and either L12 or the LS of RbuBPCase by using different serine isoacceptor tRNA species. In ,B-lactamase, the serine codon is AGU, which utilizes the serine isoacceptor species tRNASer; in L12 and the LS of RbuBPCase, the serine codewords are UCU and UCA, respectively, both of which are recognized by the serine isoacceptor species tRNA er. By using either pure tRNA1er or pure tRNA3er, the expression of each gene can be quantitated. In this system, guanosine-5'-diphosphate-3'-diphosphate inhibits the expression ofthe ,j-lactamase and L12 genes but stimulates the synthesis of the LS. In addition, the ratio of fMet-Ser/fMet-Ala (L12/L1O) synthesized was about 1 as compared with the ratio of 4 that has been obtained previously in vivo or in in vitro protein-synthesizing systems, in which the entire gene product was measured. A simplified DNA-directed coupled protein-synthesizing system has been developed to study the regulation ofthe synthesis of ribosomal protein L10 (1). In these initial experiments, plasmid pNF1337 DNA (2) was used as template, and the formation ofthe first dipeptide (fMet-Ala) ofprotein L10 was determined. An important advantage of this DNA-dependent system is that it can be constructed with only five highly purified proteins, i.e., RNA polymerase, initiation factor 1 (IF-1), IF-2, IF-3, and elongation factor Tu (EF-Tu). By using fMet-tRNA and the appropriate aminoacyl-tRNA species for the second amino acid, a specific dipeptide gene product can be selectively synthesized. Because dipeptide formation from a DNA template involves accurate transcription and proper initiation of translation, this system is ideally suited for studies on the regulation of gene expression.The ideal templates are plasmids or purified mRNAs that direct the synthesis of a limited number of products with different second amino acids. Plasmid pBR322 and recombinant plasmids derived from pBR322 contain the ,B-lactamase gene (3), which is efficiently expressed. Thus, the amino-terminal -fragment of ,B-lactamase (fMet-Ser) will be synthesized in the dipeptide system when fMet-tRNA and Ser-tRNA are present. A difficulty arises when a plasmid has another gene that also contains the serine codeword as the second amino acid. This is, in fact, the case with two recombinant plasmids derived from pBR322 that are described in this study. Plasmid pNF1337 contains, in addition to the fl-lactamase gene, the genes for LIO,...

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP

“…A limited in vitro translation reaction in which only the first two tRNAs are present was performed, using malEA2-8 and malEA2-8supl mRNAs as substrates. The rate of synthesis of the 2-amino-acid initiation peptide fMetIle was then determined as previously described (9,24,30) (Table 2). Results from this analysis revealed that the rate of translation of the initiation peptide from malEA2-8supl mRNA was approximately 18 times higher than from malEA2-8 mRNA.…”

“…In vitro transcription of the malE gene was carried out as described previously (32). In vitro translation and quantitation of the rate of synthesis of the 2-amino-acid initiation peptide Met-Ile from malEA2-8 and malEA2-8supl mRNAs was performed essentially as described by Weissbach and coworkers (9,24,30). The isolation of 70S initiation complexes was carried out as described previously (29).…”

Nucleotide sequence and expression of the aceK gene coding for isocitrate dehydrogenase kinase/phosphatase in Escherichia coli

“…An identical procedure was followed for constructing the recombinant plasmids pBC303.480 and pBC303.481, except that gene fusion was achieved in a different translational phasing. translation coupled system (6). In this system, plasmiddirected protein synthesis is limited to the formation of the N-terminal dipeptide of the gene product instead of the completed protein.…”

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

“…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)(2)(3)(4)(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).…”

“…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).…”