Structure and Properties of an RNA Primer for Initiation of Rous Sarcoma Virus DNA Synthesis In Vitro

Abstract

An early, obligatory step in replication of RNA tumor viruses is transcription of the genetic information of the virion RNA genome into DNA. This DNA is then integrated as the provirus into the DNA of the host cell (Temin 1964(Temin , 1971 Varmus et al., this volume). Like all DNA polymerases studied so far, the virion-associated RNA-directed DNA polymerase of Rous sarcoma virus (RSV) requires a nucleic acid primer molecule to initiate synthesis (Fliigel and Wells 1972;Verma et al. 1972;Taylor et al. 1973; Fltigel et al. 1973).The primer which is used by the RSV DNA polymerase, at least in vitro, has been shown to be a small RNA (Canaani and Duesberg 1972;Bishop et al. 1973; F a r a s et al. 1973b). This 4S RNA primer is normally associated with the high molecular weight genomic RNA but can be removed by den a t u r i n g the complex.Since 15-30% of the RNA in virions of RSV is small (4-7S) (Robinson et al. 1965;Duesberg 1968; Bishop et al. 1970a,b;Sawyer and Dahlberg 1973; F a r a s et al. 197.3a,b), we asked whether these RNAs were a specific class of molecules, r a t h e r t h a n just degradation products of high molecular weight RNA; our d a t a indicated that they were specific. We then studied one species of 4S RNA in detail, an RNA which serves as primer for RSV DNA synthesis in vitro. In this report we review and extend our previous studies on this RNA. therefore feel that the molecules which are being fractionated are specific and not just random degradation products of high molecular weight RNA. We do, however, occasionally note some haze in the gel pattern, which probably reflects degradation products in some RNA preparations.The purity of the m a t e r i a l in each spot on the twodimensional gel was ascertained by elution of the RNA from the gel, digestion with RNase TI or pancreatic RNase and two-dimensional fractionation RSV Virion RNAsIn order to study the possible functions of individual small RNA species of RSV virions, we fractionated them by two-dimensional poiyacrylamide gel electrophoresis (Ikemura and Dahlberg 1973;Sawyer and Dahlberg 1973). This procedure is very rapid and well suited for work using low amounts of 32p-labeled RNAs. When applied to the small RNAs of RSV, the method resolved the mixture into at least ten separate fractions, each of which was visualized as a spot on an autoradiogram of the gel (Fig. 1).This RSV small RNA gel pattern has been reproduced more than 15 times in our laboratory. We * Present address: The Rockefeller University, Figure 1. Two-dimensional polyacrylamide gel fractionation of total RSV small RNAs. Total RNA was prepared from purified 32p-labeled virions of the Schmidt-Ruppin-D strain of Rous sarcoma virus by pronase treatment, phenol-SDS extraction and ethanol precipitation (Altaner and Temin 1970;Davis and Rueckert 1972;Sawyer and Dahlberg 1973). The precipitated RNA was dissolved in 50 ~1 0.045 M Tris, 0.045 M boric acid, 0.0014 M EDTA pH 8.3 saturated with urea (at 5~ heated to 95~ for 2 min, mixed with 50 Izl melted 2% agarose in the same buff...

Genetics of RNA Tumor Viruses

“…Among those 4 S RNA species bound to the 60-70 S RNA is one which serves as a primer for RNA-dependent DNA synthesis in vitro (Canaani and Duesberg, 1972). Its structure has been determined; it is identical to a cellular tryptophan transfer RNA species and shows specific binding affinity to RNA-dependent DNA polymerase Dahlberg et al, 1974Dahlberg et al, , 1975Panet et al, 1975b). This 4 S RNA species is the only low molecular weight RNA species in the virion for which a function is known.…”

Immunochemical Studies of Beef Pancreas Tryptophanyl‐tRNA Synthetase and Its Fragments

“…yeast tRNATrp, and the sequences of tRNAsTrp from beef and chicken are alike [22,22]. Chicken tRNATrp is known to be a primer for reverse transcriptase from avian myeloblastosis virus 1221 ; moreover, reverse transcriptase forms in vitro a complex with chicken and beef tRNAsTrP [23-251. Therefore, one may presume that tryptophanyl-tRNA synthetase and reverse transcriptase possess some regions of their structure in common.…”

“…Yeast tRNATrP differs in structure from primer tRNATrp [22,26] in the region of nucleotides complementary to viral RNA [27], and cannot serve as a primer. It was of interest to elucidate whether yeast tRNAT'p can bind to reverse transcriptase.…”

5.9‐S RNA, a New RNA Characterized in Several Mammalian Cell Lines

“…It is generally accepted that all these RNAs are incorporated passively into the virions at the time of 'budding', and thus may have no special function. However, more recently, it has been shown that the synthesis of cDNA from 70-S RNA by reverse transcriptase uses an RNA primer which has been identified as tryptophan-tRNA in the avian system [7,8].In the course of an investigation designed to identify the RNA priming the transcription of the murine oncornavirus genome by reverse transcriptase, the presence in acrylamide gels of a band representing a new species of RNA was noted as a constant finding. This species of RNA, which we will refer to as 5.9-S RNA, is found in murine oncornaviruses, and also in cell lines, both virus-producing and non-producing.…”

“…It is generally accepted that all these RNAs are incorporated passively into the virions at the time of 'budding', and thus may have no special function. However, more recently, it has been shown that the synthesis of cDNA from 70-S RNA by reverse transcriptase uses an RNA primer which has been identified as tryptophan-tRNA in the avian system [7,8].…”