A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.
Digestion of 30S ribosomal subunits from E. coli by insoluble ribonuclease produces three subparticles. The ribosomal proteins associated with each of these particles were identified. Some of the proteins are associated with only one of the three subparticles. The protein compositions of the three particles can be arranged in an overlapping linear sequence of five groups. Furthermore, inspection of the previously determined assembly sequence of the 30S proteins indicates that proteins associated in the subparticles are interdependent in the assembly process. Several investigators have recently described conditions for mild degradation of Escherichia coli ribosomes by nucleases (1-7). In general, these workers have shown that controlled nuclease digestion can produce large subparticles from both 50S and 30S ribosomes. One conclusion developed in these experiments is that the three-dimensional structure of the native ribosome influences the susceptibility of the RNA to enzymatic attack. If this conclusion is correct, then the products of limited digestion by nucleases should reflect gross features of the three-dimensional structure of the ribosome. Thus, an analysis of the protein compositions of the particles released by nuclease action might give some clues to the threedimensional arrangement of the proteins. Brimacombe et al. (7) have recently published experiments designed with this underlying concept. They examined several isolated fragments of the 30S ribosome and found that they contained different proteins. However, they were not able to identify the proteins.We have developed conditions to digest the 30S ribosome with ribonuclease, with the aim of determining the distribution of the 21 proteins of this subunit among its resultant subparticles. Brief exposure of the 30S ribosome suspension to insoluble ribonuclease A produces three major products, sedimenting at about 22 S, 15 S, and 7 S. We have analyzed each subparticle for its protein composition. The 22S particle contains 15-16 proteins, the 15S particle has 12-13 proteins, and the 7S particle has 6-8 proteins. Each of the 21 proteins appears to be a component of at least one of the three particles. Comparison of the distribution of the proteins with their sequence of assembly, as determined by Mizushima and Nomura (8)
Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.
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