tRNA yields a product about 10-12 amino acid residues longer than the product ... added to 1 ml of protein synthesis reaction mixture to a final concentration of ...
Proc. Natl. Acad. Sci. USA Vol. 74, No. 12, pp. 5509-5513, December 1977
Biochemistry
Initiation of polypeptide synthesis with various NH2-blocked aminoacyl-tRNAs under the direction of alfalfa mosaic virus RNA 4 (capped mRNA/ribosome recognition site/heterologous translation/acetylphenylalanyl-tRNA/product characterization)
A. CASTEL, B. KRAAL, P. R. M. KERKLAAN, J. KLOK, AND L. BOSCH Department of Biochemistry, State University, P.O. Box 9505, 2300 RA Leiden, The Netherlands
Communicated by L. N. M. Duysens, October 11, 1977
ABSTRACT Initiation of polypeptide synthesis in a cell-free system of Escherichia coli directed by alfalfa mosaic virus RNA 4 was studied by using either fMet-tRNA or Ac-Phe-tRNA as initiator tRNA. Initiation.with fMet-tRNA yielded a product that was identical to the authentic viral coat protein except that the NHrterminal serine was preceded by fMet instead of being acetylated. When Ac-Phe-tRNA was used as initiator, the biosynthetic product was 10-12 amino acid residues longer, the extra amino acids being located at the NH2-terminus. fMettRNA and Ac-Phe-tRNA did not compete for ribosomes during initiation of protein synthesis, as became evident from incorporation studies using both initiator tRNAs simultaneously. It is concluded that E. coli ribosomes recognize two sites on the 5' end of alfalfa mosaic virus RNA 4 that are separated by a region of about 30 nucleotides. The results are in complete agreement with the 5'-terminal nucleotide sequence of this RNA [Koper-Zwarthoff, E. C., Lockhard, R. E., RajBhandary, U. L., Alzner-deWeerd, B. & Bol, J. F. (1977) Proc. Nati. Acad. Sci. USA 74, 5504-55081.
The primary event during initiation of polypeptide synthesis is the binding of the ribosome to a specific site on the messenger. The question of how the ribosome recognizes this site has been the subject of study in many laboratories (1-4), including our own (5-7). Several years ago we were struck by the finding (8-10) that even in a heterologous system consisting of Escherichia coli ribosomes and a plant viral messenger -this event occurs with a remarkable specificity. The messenger used was RNA derived from the top component a of alfalfa mosaic virus (AlMV), here designated AIMV RNA 4. The specificity of the interaction became apparent when the binding of 16 different species of 14C-labeled aminoacyl-tRNA to ribosome-AlMV RNA complexes was studied. Only three species, Phe-tRNA, Ile-tRNA, and Val-tRNA, could bind. Two of them responded to adjacent codons on the messenger, because binding of Ac-Phe-tRNA and Ile-tRNA resulted in the formation of Ac-Phe-Ile-tRNA. Aminoacyl-tRNA species that might respond to codons overlapping the two adjacent ones in the hexanucleotide coding for Phe and Ile did not bind. Moreover, either Ac-Phe-tRNA or Ac-Ile-tRNA could function as polypeptide chain initiator. Ac-Phe-tRNA bound to the donor site and Ac-Ile-tRNA to the acceptor site. The latter tRNA could be translocated to the donor site by adding GTP. Furthermore, chains were initiated in the usual way by fMet-tRNA. These results suggested the existence of at least two E. coli ribosome binding sites on AIMV RNA 4, which were possibly overlapping, because only one ribosome per RNA molecule could be bound at the same time. This might indicate that the four triplets coding for Phe, Ile, Val, and fMet are all located in one stretch of 30-40 nucleotides. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
In the present paper we show that initiation with Ac-PhetRNA yields a product about 10-12 amino acid residues longer than the product initiated with fMet-tRNA. This result is in striking agreement with the 5'-terminal nucleotide sequence reported by Koper-Zwarthoff et al. (11): 1
4
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m7GpppGUU-UUU-AUU-.. .-AUG-AGU-UCU-UCA-.. Val- Phe - Ile -. . .-Met - Ser - Ser - Ser
The nucleotide sequence shows that the triplets coding for Val-, Phe-, and Ile-tRNA are located right after the cap and in phase with the coat protein cistron. Initiation with Ac-PhetRNA and with fMet-tRNA appeared not to compete with each other, indicating that the E. coli ribosome makes its choice between the two sites on this viral RNA before the binding of either initiator tRNA. MATERIALS AND METHODS Isolation of AIMV RNA and AIMV Coat Protein. Nucleoprotein material from AMV 425 was isolated from tobacco (12) and separated into the four main components (13). RNA was extracted and purified by centrifugation in an MSE zonal rotor. The preparations were analyzed on 3% polyacrylamide gels' (14). AlMV coat protein was prepared by dissociating the virus with 0.05 M MgCl2 (15). Preparation of E. coli Cell-Free Extracts and In Vitro Protein Synthesis. Cell-free extracts of E. coli MRE 600 were' prepared according to the procedure of Nirenberg and Matthaei (16), slightly modified by Voorma et al. (17). The reaction mixture for protein synthesis (total volume, 100,l) contained 30 ,ul of S30 extract (30,000 X g supernatant), 9 ,ug of leucovorin, 1 mM phosphoenolpyruvate, 0.12 mM GTP, 0.05 mM each unlabeled amino acids, 12 mM KCI, 50 mM Tris-HCI (pH 7.8), 5 mM 2-mercaptoethanol, 13 mM MgCl2, viral RNA and labeled amino acids as indicated in the legends. After a 30-min incubation at 370 the reaction mixture was treated with 0.1 ml of 0.1 M NaOH at 370 for 15 min. Polyacrylamide Gel Electrophoresis of Biosynthetic Products. When labeled biosynthetic polypeptides had to be analyzed by polyacrylamide gel electrophoresis, 3 ml of 7% (wt/vol) trichloroacetic acid was added to the reaction mixture after alkali treatment. The pellet was washed with the same solution, rinsed with acetone, and dried. The pellet was taken up into sample buffer (100 Mi) containing 1% (wt/vol) sodium dodecyl sulfate (NaDodSO4) and 10% (vol/vol) 2-mercaptoethanol, and the mixture was heated in a boiling-water bath for 5 min. Analysis occurred on 10% polyacrylamide gels containing 0.1% (wt/vol) NaDodSO4 as previously described (18). Ge1g were -sliced into 1-mm- sections, which were placed in Abbreviations: AIMV, alfalfa mosaic virus; BMV, brome mosaic virus;
NaDodSO4, sodium dodecyl sulfate. 5509
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Biochemistry:
Castel et al.
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