Fidelity of translation of satellite tobacco necrosis virus ribonucleic acid in a cell-free Escherichia coli system.

Abstract

Biochemistry @ Copyright 1973 by the American Chemical Society Volume 12, Number 2 January 16,1973 Fidelity of Translation of Satellite Tobacco Necrosis Virus Ribonucleic Acid in a Cell-Free Escherichia coli Systemt R. Rice* and H. Fraenkel-Conrat The polypeptides synthesized in an E. coli cell-free system under the direction of the small RNA of the satellite tobacco necrosis virus were compared with the authentic coat protein of this virus. Many of the tryptic peptides obtained from the two types of proteins moved identically upon high- resolution cation exchange column chromatography, but distinct qualitative and quantitative differences between the two peptide patterns were also evident. Gel electrophoresis in 9 M urea at pH 4.0 showed that the biosynthesized material was not homogeneous in regard to charge; some of it showed a ABSTRACT: E scherichia coli cell-free systems synthesize recognizable proteins when programmed with messengers from the small RNA bacteriophages (Stavis and August, 1970). These extracts generally respond better to exogenous messenger RNA, particularly in quantitative respects, than do incorporation systems from plant and animal sources. The genetic code, possibly including initiation (Stewart et at., 1971 ; Housman et a/., 1970; Marcus et a/., 1970) and termination (Goldstein et al., 1970) codons, seems to be identical in the animal, plant, and bacterial kingdoms. Hence, E. coli extracts have been used with animal and plant viral RNA messengers to investi- gate translation in these heterologous systems and to study the viral gene products in vitro. The most direct test to establish whether accurate translation of a viral messenger occurs has been to ascertain whether the viral coat protein is synthesized in vitro since other gene prod- ucts have not been as readily identifiable. Some studies have indicated considerable similarity between translation products and viral coat protein. For example, with the RNA from the t From the Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720. Receired August 18, 1972. This investigation was supported by U. S. Public Health Service Grant No. G M 01389 from the National Institute of General Medical Sciences, and Grant No. GB 6209 from the National Science Foundation. R . Rice was a recipient of a National Science Foundation Graduate Fellowship. * Address correspondence to : Institute of Marine Resources, Uni- versity of California, Davis, Calif. 95616. mobility similar to that of the authentic coat protein. Gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of a wide range of molecular weight species in the biosynthesized protein ; the largest, representing about 5 % of the total, coincided with authentic virus coat pro- tein. It is concluded that much of the information on satellite tobacco necrosis virus RNA is translated into proper amino acid sequences in the E. coli system, but that only very little complete coat protein, and that probably carrying a terminal formyl-methionyl group, is synthesized. satellite of tobacco necrosis virus (STNV)' (Clark et a/., 1965) and the similarly small top a component of alfalfa mosaic virus (van Ravenswaay Classen et a/,, 1967), two-dimensional peptide maps have been reported as showing nearly complete coincidence of radioactive spots from the translation products and ninhydrin-positive spots from the coat proteins. In con- trast, earlier studies with TMV-RNA had indicated no clear evidence for coat-related material being synthesized in citro (Aach et a/., 1964; Schwartz, 1967). Studies with brome mosaic virus RNA (Stubbs and Kaesberg, 1967) and poliomyelitis virus RNA (Rekosh et al., 1970) gave somewhat ambiguous results and the recent claim that avian myeloblastosis virus RNA directs the synthesis of viral antigens (Siegert et a/., 1972) must await substantiation. In view of these widely varying results, it was our intention to reinvestigate one of the reports indicating positive results. We chose to characterize the translation products of STNV- RNA using more discriminating techniques which have be- come available since the original report appeared. The RNA of STNV is a convenient messenger for this kind of work in that it is small (0.4 X lo6) and can code maximally for only one small protein in addition to the viral coat protein (Roy et at., 1969). Since the completion of this work, an interesting study of the translation of STNV-RNA in E. coli and wheat embryo 1 Abbreviations used are: STNV, satellite tobacco necrosis virus; TMV, tobacco mosaic virus; TPCK, ~-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone. BIOCHEMISTRY V O , L . 1 2 , NO.