Coli Cell-free System Research Articles

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [ 35S]methionine-containing chymotryptic peptides of the 52,000 M r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [ 35S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.

Translation of AKR-murine leukemia viral RNA in an [formula omitted] cell-free system

High molecular weight RNA isolated from the oncogenic type C murine leukemia virus, AKR-MuLV, stimulates the incorporation of radioactive amino acids into protein in an E. coli cell-free system. Analysis of the translational products by SDS polyacrylamide gel electrophoresis demonstrated the synthesis of at least three proteins corresponding in molecular weight to several authentic viral proteins. Positive immunoprecipitation tests also confirm the translational product as AKR-MuLV related. Although at least 18 proteins were found on analysis of disrupted murine leukemia virions, only three were synthesized in vitro in response to AKR-MuLV RNA in the E. coli cell-free system.

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

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.

Translation of oncogenic viral RNA and eukaryotic messenger RNA in the E. coli cell-free system

Translation of oncogenic viral RNA and eukaryotic messenger RNA in the E. coli cell-free system

Protein synthesis in Escherichia coli extracts programmed by poliovirus RNA

Poliovirus RNA is as active in the stimulation of amino-acid incorporation in an Escherichia, coli cell-free system as is an equimolar amount of f2 phage RNA. The tryptic peptides of the product specified by poliovirus RNA correspond to tryptic peptides from authentic poliovirus protein, although not every authentic tryptic peptide is found in the in vitro product. Initiation of protein synthesis occurs at eight or more distinct places on poliovirus RNA and each initiated polypeptide starts with N-formylmethionine. Initiation is maximal at a concentration of 8 m m-magnesium ion and requires the E. coli initiation factors. Polyacrylamide gel analysis of the product in the presence of sodium dodecyl sulfate, shows that the product is a heterogeneous mixture of polypeptides. The largest polypeptide formed is about 60,000 daltons. Analysis on sucrose gradients of the RNA incubated in the E. coli cell-free system indicates that the RNA is rapidly degraded. This degradation seems to be, at least in part, responsible for the limited size and heterogeneity of the product.

Inhibition of polypeptide synthesis in E. Coli cell free system by influenza viral protein

Inhibition of polypeptide synthesis in E. Coli cell free system by influenza viral protein

Rapidly-labelled ribonucleic acid-protein complexes of the thyroid tissue.

The existence of non-ribosomal rapidly-labelled ribonucleoprotein particles has been examined by isopycnic and sucrose gradient centrifugation in nuclear and cytoplasmic subcellular fractions of sheep thyroid tissue. These particles have been described in the saline extract (pH 8.0) of purified nuclei, in EDTA-treated polyribosomes and in postribosomal regions of deoxycholatetreated postmitochondrial supernatant. They have been identified as a special class of ribonucleoproteins rich in proteins by the following criteria: low buoyant densities in CsCl gradients (from 1.36 to 1.41 g/ml, with a predominance at 1.41), DNA-like base composition of their constitutive RNA, increase or decrease of their buoyant densities after trypsin or ribonuclease digestion. Although the specific nature of the constitutive protein(s) is not yet determined, it appears that both nuclei-associated and cytoplasmic (postribosomal and polyribosomal) rapidlylabelled ribonucleoproteins are complexes of messenger-like RNA and protein in a proportion of about 15 to 30% RNA, 85 to 70% protein by weight. RNA of cytoplasmic postribosomal ribonucleoprotein particles is heterogeneous and composed of species ranging from about 6 S to 22 S. It stimulates protein synthesis in the E. coli cell-free system whereas the corresponding ribonucleoproteins are poorly stimulatory. The relationships between these particles and their possible role in messenger RNA function are discussed.

Translation of poliovirus RNA by an E. coli cell-free system.

Poliovirus RNA is an ideal mammalian mRNA for in vitro studies of protein biosynthesis because it can be prepared in fairly large quantities in an undegraded form from virions. It is known to direct the synthesis of several poliovirus proteins in infected HeLa cells. The tryptic peptides of these proteins can be identified (Jacobson, Asso, and Baltimore, in prep.; Baltimore, Jacobson, Asso, and Huang, this volume) and compared with those made in vitro. In vivo, it apparently codes for only a single polypeptide which is subsequently cleaved to form the functional proteins (Jacobson and Baltimore, 1968b; Baltimore et al., this vol.).

Function of reticulocyte ribonucleic acid in the Escherichia coli cell-free system

1. 1. The addition of reticulocyte RNA to an Escherichia coli cell-free system results in the enhancement of amino acid incorporation into protein. The protein synthesized was digested with trypsin (EC 3.4.4.4) and the resulting peptides compared with the tryptic peptides of hemoglobin. There is no correspondence between the two sets of peptides under the conditions described. 2. 2. There is a marked correspondence, however, between the tryptic peptides synthesized in the E. coli cell-free system alone and those synthesized in the reticulocyte RNA enhanced E. coli system. This suggests that the added reticulocyte RNA is acting to promote or protect the endogenous activity of the cell-free system.

Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system.

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system. J. Bacteriol. 92:197-203. 1966.-When tetracycline, an inhibitor of cell-free protein synthesis, was preincubated with each component of the Escherichia coli cell-free system, i.e., ribosomes, soluble ribonucleic acid (sRNA), polyuridylic acid (poly U), and S-100 (supernatant enzymes), only the ribosomal-bound antibiotic was inhibitory to the cell-free assay. Experiments designed to further localize the site of inhibition to either the 50S (Svedberg) or the 30S ribosomal subunit were not conclusive. Tritiated tetracycline (7-H(3)-tetracycline) was bound to isolated 50S ribosomes, and these were recombined with 30S subunits to form 70S ribosomes. When these ribosomes were dissociated and the subunits reisolated, the antibiotic was found with both the 50S and the 30S particles. The same results were observed when the tetracycline was initially bound to the 30S subunit.

Polyribonucleotide-directed protein synthesis using an E. coli cell-free system.

SINCE the discovery by Nirenberg and Matthaei1 that a synthetic polynucleotide, when added to an E. coli cell-free system, led to the synthesis of a specific polypeptide, various groups have used similar methods to find code letters, for the common amino-acids. We should like to report certain observations, some of which are at variance with those already published.