Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus

Abstract

The structure and molecular weight of the 60-70S RNA complex and the 30-40S RNA species of Rous sarcoma virus were analyzed in an electron microscope after treatment of the RNAs with the bacteriophage T4 gene-32 protein to stretch out the RNA strands. Although all RNA preparations treated with gene-32 protein showed considerable heterogeneity in length, a significant fraction of the RNA retained its original sedimentation coefficient after treatment to allow the following conclusions to be made: The 30-40S RNA was confirmed to be a linear polynucleotide with a molecular weight of about 3 X 106. The 60-70S RNA exhibited a network structure with a molecular weight predominantly of about 6 X 106. Therefore, the subunit hypothesis for the 60-70S RNA is confirmed. A model for the structure and molecular weight of the 60-70S RNA postulates that the complex consists of two 30-40S RNA subunits held together at many points. This model elucidates the biological observation that the infectivity of RNA tumor viruses is proportional to the amount of 30-40S RNA in a virus preparation and not to the amount of 60-70S RNA.The structure and the molecular weight of tumor virus RNA have been difficult to study experimentally. The molecular weight is thought to range between a minimum of 3 X 106 and a maximum of 12 X 106 (1-10). This uncertainty is due mainly to the complex structure of the viral RNA (3) and to the difficulty in determining the exact content of RNA in purified virions (10). Extraction of the nucleic acid from virions followed by sedimentation reveals a major RNA species with a sedimentation coefficient of 60-70 S. When this RNA is placed in an environment wherein its secondary structure should be destroyed, the sedimentation coefficient decreases to 30-40 S and, at the same time, its electrophoretic mobility in sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels increases about 2-fold (11). The molecular weight of the 30-40S RNA species has been determined under denaturing conditions, and is about 3 X 106 (11). Thus, the 60-70S RNA was thought to be segmented, containing two, three, or four 30-40S RNA subunits held together by basepairing involving short complementary sequences.Although there is good, indirect, evidence for a subunit structure of the 60-70S RNA, nothing is known about how these subunits are linked. Linkage among 30-40S RNA subunits may be at only one site between two subunits, compatible with a relatively extended 60-70S RNA complex; or it may occur at several sites, compatible with a more compact, network-type 60-70S RNA complex. Furthermore, the available evidence does not exclude unequivocally that the conversion of tumor virus RNA from 60-70S to 30-40S may be due to an extreme conformational change rather than to a dissociation into subunits. Although there is no precedent for a conformational change that decreases the sedimentation coefficient but increases the electrophoretic mobility of nucleic acids, it was desirable to obtain independent evidence for the postulated subunit structu...

Preliminary Physical Mapping of RNA-RNA Linkages in the Genomic RNA of Moloney Murine Leukemia Virus

“…Since the earliest investigations of retroviral RNAs (15), it has frequently been observed that dimeric RNAs appear intact even when they are composed of stochastically fragmented monomers (18). This observation shows that viral RNAs contain linkages other than the primary or most stable dimeric linkage but does not allow one to draw any inferences concerning the locations of these secondary linkages.…”

Retroviral RNA dimer linkage.

“…Under less stringent conditions (Mangel et al, 1974) multiple points of contact have been noted, suggesting that the single linkage observed under more restrictive conditions might be a primary association or dimerization initiation site (DIS). Recent work from Ho$ glund et al (1997), looking at the dimer linkage of HIV-1 showed, interestingly, that there did not appear to be any free 5h ends, indicating that the two RNAs were interacting in addition at site(s) upstream of the recognized DLS.…”

Stabilities of Avian Sarcoma Virus RNAs: Comparison of Subgenomic and Genomic Species with Cellular mRNAs

“…The structure of the mRNA for the putative precursor to the reverse transcriptase Prl80gag -p°~ has not yet been established, but information derived from the 0022-1317/83/0000-5704 $02.00 © 1983 SGM sequence of the Prague C strain genome RNA suggests the possibility that it is coded for by an approximately 9.3 kb RNA in which the termination codon for Pr76 ga0 is removed by splicing (D. Schwartz, personal communication). Besides these RNA species, additional 9.3 kb RNA molecules are packaged into virions as a dimeric 70S complex (Mangel et al, 1974). It is not yet clear whether the 9.3 kb RNA that is packaged is identical to the 9-3 kb mRNA.…”