Translation of satellite tobacco necrosis virus ribonucleic acid. II. Initiation of in vitro translation in procaryotic and eucaryotic systems.

This paper reports the N-terminal amino acid of the product of in vitro translation of satellite tobacco necrosis virus ribonucleic acid (STNV-RNA) by both a procaryotic (Escherichia coli) and eucaryotic (wheat embryo) system. In vitro translation of satellite tobacco necrosis virus RNA by the procaryotic (Escherichia coli) system initiates with fMet-tRNAyet. Specifically, deformylation of the in vitro product protein followed by end-group analysis with fluorodinitrobenzene reveals DNP-Met. At 6-7 mM Mg2+ levels, extracts from Escherichia coli deprived of formyl donors by the action of trimethoprim require formyltetrahydrofolic acid for translation of the RNA. The viral-RNA-dependent incorporation of iaH]formate from [ 3H]formyltetrahydroP rotein chain initiation in procaryotes (Marcker and Sanger, 1965; Adams and Cappechi, 1966; Eisenstadt and Lengyel, 1966; Horikoshi and Doi, 1968), and presumedly mitochondria and chloroplasts of higher organisms (Smith and Marcker, 1968; Galper and Darnell, 1969), utilizes the codon AUG (Thach et al., 1966) as a chain-initiation signal to code for fMet-tRNA?. The resultant initial product t From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801. Receiued December 29, 1971. Supported in part by National Institutes of Health Research Grant GM-08647. t Present address: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, N. Y . 10461. Abbreviations used are: STNV, satellite tobacco necrosis virus; TYMV, turnip yellow mosaic virus; fMet, N-formylmethionine; fH4Fo1, "0-formyltetrahydrofolic acid; tRNAYet, methionine-specific-tRNA capable of accepting a formyl group on its methionine; Met-tRNAFet, rnethionyl ester containing tRNAflet; fMet-tRNAyet, formylmethionyl containing tRNAyet; tRNAYet, methionine-specific initiator tRNA not capable of accepting a formyl group on its methionine; MettRNA:"', methionyl ester containing tRNA?; FDNB, Auorodinitrobenzene. 2014 B I O C H E M I S T R Y , V O L . 1 1 , N O . 1 1 , 1 9 7 2 folic acid into protein results in the preferential labeling of one tryptic fingerprint peptide. Digestion of the in vitro product protein with specific proteases followed by ion-exchange procedures yields N-formylmethionine. Similar ion-exchange analyses of the product of in vitro translation of STNV-RNA by the eucaryotic (wheat embryo) system fail to detect M e t in the product. In contrast, end-group analyses of the in vitro eucaryotic product reveal Ala as the most prevalent N-terminal amino acid. These data support the theory that the original, eucaryotic, STNV-RNA translation product has specifically lost an N-terminal methionine to yield an alanine-terminated protein. proteins of such systems, containing formylmethionine in their N termini, are then modified, if necessary, by deformylation and/or exopeptidase action, to yield the final product proteins (Takeda and Webster, 1968). The mechanism of protein chain initiation in the cytoplasmic fraction of cells of higher organisms (eucaryotes) is not as well characterized. Current evidence indicates that a nonformylated, yet methionine-specific tRNA is uniquely involved in protein chain initiation in the cytoplasm of eucaryotic systems (Smith and Marcker, 1970; Marcus et al., 1970; Housman et al., 1970; Wigle and Dixon, 1970). Other evidence suggests that a deacylated, possibly methioninespecific, tRNA is involved in this initiation process (Culp et al., 1970). A role for some form of methionine-specific tRNA is central to all of these initiation schemes. Thus, the methionine codon AUG is presumedly involved. Yet protein chain initiation must require a mechanism more complex than the existence of the methionine codon sequence, AUG. Specifically, protein chain initiation must avoid "missense" initiations T R A N S L A T I O N O F P L A N T V I R U S R N A spanning portions of two consecutive codons. Further, protein synthesis must distinguish between AUG sequences that code for protein chain-initiation positions and AUG sequences that code for internal methionine residues. Satellite tobacco necrosis virus RNA provides an ideal probe to investigate these subtleties of protein chain initiation. First, STNV-RNA is a small, possibly monocistronic message for the viral coat protein (Reichmann, 1964). Second, STNV-RNA serves as a functional message in cellfree systems from both procaryotic (Escherichia coli) and eucaryotic (wheat embryo) sources (Clark et al., 1965; Klein et a[., 1972). Further, typtic digestion of the products produced from these two in vitro systems, followed by fingerprint analysis, reveals coincidence between the detectable radioactive peptides and a majority of the tryptic peptides derived from STNV coat protein (Klein et al., 1972). Thus, within the limits of detection by fingerprint analysis, translation of STNV-RNA by both the procaryotic and eucaryotic systems is correct and essentially complete. These convenient features emphasize the need for a closer examination of the initiation of translation of STNV-RNA. This paper reports on the initial amino acids of the STNVRNA translation product made by in vitro procaryotic and eucaryotic systems. Experimental Procedures Materials. Cell-free extracts of E . coli A-19 were prepared from cells grown and extracted as previously described (Clark et al . , 1965). Extracts of E. coli A-19 deficient in formyl components were prepared by trimethoprim treatment (Eisenstadt and Lengyel, 1966). The cell-free eucaryotic system employed s-23 extracts of wheat embryo (Marcus et al . , 1968). STNV-RNA was obtained from STNV by phenol extraction (Kirby, 1965) of virus grown on tobacco (Reichmann, 1964) or mung beans (Liu et al., 1969). The STNV-RNAs obtained from virus isolated from these varied sources are identical in physical properties and yield identical translation products upon fingerprint analysis. TYMV-RNA was obtained by phenol extraction (Kirby, 1965) of TYMV grown on Chinese cabbage. [ aH]Formyltetrahydrofolic acid of specific activity of 1.27 Ci/mmole was prepared enzymatically from tetrahydrofolic acid (Goldthwait and Greenberg, 1955). Sodium [ 3H]formate was purchased from Tracerlab, Co., Waltham, Mass. [14C]Amino acids and amino acid mixtures were as sold by New England Nuclear Co. Trimethoprim was the generous gift of Dr. G. H. Hitchings of Burroughs Wellcome Co., Tuckahoe, N. Y. Specific enzymes were purchased from Worthington Biochemical Corp., Freehold, N. J . fMet was purchased from Mann Research Laboratories, New York, N. Y. fMet-Ala-Lys was the generous gift of Dr. Phillip Leder, NIH, Bethesda, Md. Enzyme Incubations. Unless stated otherwise, reaction conditions for the cell-free E . coli assays were as previously reported (Klein et al . , 1972) with the following exceptions. Where listed, certain reactions involved Mgz+ concentrations other than 0.01 M Mgz+. As listed, certain reactions include 50 p~ ~[ l~C]Met (specific activity of 100-150 mCi/ mmole) supplemented with 40 p~ concentrations of the 19 other L-[' ZC]amino acids required for protein synthesis. As indicated, certain reactions contained 40 p~ [ 3H]fH4Fol (specific activity 1.27 Ci/mmole) supplemented with 40 p~ levels of the 20 ~-[1zC]amino acids found in proteins. The 20-min, 37" incubations employed are sufficient to allow all reactions to go to completion. The incubation conditions for assays with the cell-free wheat embryo system were as reported (Klein et al., 1972) except that the incorporation utilized single [3H]or [l4CC]amino acids supplemented with 3 X M concentrations of the other 19 ~ [ ~ ~ C ] a m i n o acids commonly found in proteins. The 30-min, 30' incubations employed are sufficient to allow all reactions to go to completion. Analysis Methods. [ 4C]Amino acid incorporation into protein was assayed by means of scintillation counting of acid-precipitable material collected on Millipore-type HAWP filters after treatment with hot 5 % trichloroacetic acid (Conway and Lipmann, 1964). Assays involving [ 3H]formyl incorporation from [ 3H]formyl-tetrahydrofolic acid into protein were terminated by 10-min, 37' incubations in the presence of 100 pg of pancreatic RNase followed by four repetitive washings onto Millipore filters with cold (0-2') 5% trichloroacetic acid. Precipitated labeled protein retained on Millipore filters after drying at 27' for 8-12 hr was counted by liquid scintillation counting in either a Packard Model EX-314 or a Beckman Model LS-133 scintillation counter. Reactions destined for derivatization with FDNB were terminated with pacreatic RNase, as above, adjusted to pH 11 with KOH, dialyzed against 1 % (NH4)*C03 and then water until no label was detected in the dialysate, and finally freeze-dried prior to FDNB derivatization and subsequent protein hydrolysis (Fraenkel-Conrat et al., 1955). Where indicated, the existence of individual labeled amino acids in the N-terminal position of the protein product was detected by ether extraction (6 extractions of 3 ml each) of the diluted (1 M HCl) acid hydrolysate of the derivatized protein and subsequent scintillation counting of the ether extract. As indicated for other experiments, the ether extract of the acid hydrolysis mixture was analyzed by two-dimensional paper chromatography (Fraenkel-Conrat et al., 1955) along with DNP-amino acid standards. Paper sectors containing the appropriate DNP-amino acid standards were counted directly by scintillation counting without correction for localized quenching on the paper by the yellow standards. Label coincident with DNP-Met, DNP-Met sulfoxide, and DNPMet sulfone was summed and defined as DNP-Met. Where indicated, deformylation of the protein (prior to derivatization with FDNB) was carried out by 12-hr, 30' incubation of dialyzed and freeze-dried samples in 0.5 M HCl in anhydrous CH30H. Reaction mixtures destined for digestion with trypsin and carboxypeptidases A and B were terminated, titrated, dialyzed, and lyophilized as the samples prepared for FDNB derivatization. The samples were then each suspended in 1.5 ml of 0.1 M Tris-C1-0.02 M CaClZ (pH 8.3) and incubated (37') with 0.1 mg of trypsin for 8 hr before addition of another 0.1 mg of trypsin and an additional 10-12 hr of 37' incubation. After 5 min of 100' treatment to denature residual trypsin, 0.5 ml of 0.4 M NaC1-0.1 M Tris-C1-0.02 M CaC12 (pH 8.3)100 pg of solubilized carboxypeptidase A-40 pg of carboxypeptidase B were added to each sample and incubations was continued for 8 hr at 37". The samples were then stored at -15" prior to passage over a 1.2 X 15 cm Dowex 50-X2 (H+ form) column with water elution (flow rate 1 mlimin). Material adhering to the column is subsequently eluted (1 ml/min) with 0.3 M NHIOH. The deformylation of fMet was measured by ninhydrin reaction before and after varying 30" treatments with 0.5 M HC1 in anhydrous CHIOH or 1.0 M HC1 in H20. Tryptic B I O C H E M I S T R Y , V O L . 1 1 , N O . 1 1 , 1 9 7 2 2015