PERSPECTIVES AND SUMMARY ..... ... .. ..... . ..... ....... .. .. ... .. 526 THE ENZYMATIC MACHINERY: Q,B·REPLICASE AND HOST FACTOR .... 527 Identification of the Host Components and their Function in Uninftcted Escherichia coli 527 Subunit I 527 Subunits III and IV 528 The host flU:tor 529 Structure of the Replicase .... .... . .. ... .. .. .. .... ... .. .. .. .. ... . 529 Enyme shape .. . , 529 Subunit subcomplexes . ... .... .. .. .. . . .. ........ .. . . ..... .... .. ... ....... ...... .... .. . ..... .... 530 Q/J.replicase·host factor associotion ..... .. ..... ... ... ....... .. ... ...... ... .. ..... .... ... .. .. .. . .. .. . ... 531 THE GENERAL SCHEME OF IN VITRO Q/3 RNA REPLICATION ......... .. ... . 532 Plus Strands and Minus Strands .. ..... ....... . .. ........ ........ ... ......... ... ... .. .. ...... .. ... ....... . 532 Replicating RNA . .. ... . ... ... .. ... .. . . . . .... .... .. .... ... .... ..... ... . .. .. .. ... .... 532 THE ENZYMATIC MECHANISM OF IN VITRO Q/3 RNA REPLICATION .. 533 Temp/ate Specificity .. ... .. . ... . . .. .... ..... .. ... . .. . .... ........ ...... ...... ..... . .... . ..... . .. ... ..... 533 Overcoming Template Specificity .... .... ... .... . .. .. .. ... .. .. ... .. .. . ... .... .... ... .... .. .. ........ 534 Recognition of Synthetic RNAs ........ ... ... .. . ... ...... ... ...... ...... ........ .. ... . .. .. ...... ... .. .... 535

RNA replication: function and structure of Qbeta-replicase.

A factor fraction essential for the in vitro synthesis of RNA bacteriophage Qβ-RNA has been isolated from both infected and uninfected E. coli. This indicates that the contribution of the last bacterium to the replication of Qβ-RNA is more extensive than was earlier thought.

Factor Fraction required for the Synthesis of Bacteriophage Qβ-RNA

Bacterial proteins required for replication of phage Q ribonucleic acid. Pruification and properties of host factor I, a ribonucleic acid-binding protein.

The RNA replicating enzyme induced by RNA phage Qβ consists of a single phage specific and three host specific polypeptide chains. At least one of the host subunits is shown to be essential for enzymatic activity, and none are known subunits of E. coli DNA-dependent RNA polymerase.

Characterization of the subunits of Q-beta replicase.

The hypercycle - coupling of rna and protein-biosynthesis in the infection cycle of an rna bacteriophage

Synthesis of bacteriophage Q-beta RNA.

A prominent feature of the reaction catalyzed by the Qj RNA polymerase, an enzyme isolated from Escherichia coli infected with the RNA phage Qo, is the specificity for template RNA. As was first shown by Haruna and Spiegelman,' the reaction will proceed with RNA derived from the phage Qf, but not with RNA from several other natural sources. Although this requirement for Qj RNA inay be explained by the premise that discrimination between host RNA and viral RNA is essential for phage replication, such a property is, nevertheless, unusual. For example, specificity of this type is not shown by either the DNA polymerase or the DNA-dependent RNA polymerase of E. coli, both enzymes being active in vitro with a wide variety of nucleic acid preparations of both natural and synthetic sources.2-4 Furthermore, such reliance on a specific template RNA was not demonstrated by the RNA-dependent enzyme found in extracts of E. coli infected by mutant phage f2 su-11.5 Template specificity may be related to any one of three possible steps in RNA syllthesis: (a) the association of RNA and enzyme, (b) the initiation of synthesis, and (c) chain elongation.6 Evidence against exclusive binding of the enzyme to Q,B RNA has come from studies of the inhibition of the reaction by several other types of RNA and polynucleotides.7 Rather, as we previously suggested, the specificity for template appears also to involve the initiation of synthesis.8 Guanosine 5'triphosphate (GTP) has been identified as the predominant 5'-terminus of RNA synthesized in the reaction and there appeared to be only a single initiation site per molecule of template RNA. Initiation of synthesis was not detected when RNA isolated from other sources was substituted for Q,3RNA.' Further insight into this problem has now been obtained by the discovery that the Qf RNA polymerase will utilize certain synthetic polynucleotides as template. As with Q3 RNA, the specificity of template function with synthetic polymers appears to be related to the initiation of synthesis with GTP. These findings are the subject of the present report. Methods.-Preparation of enzymes: The Q3 RNA polymerase was purified from extracts of Qf10infected E. coli Q13.11 Frozen infected cells, 50 gm, were ground with 100 gm alumina and extracted with 200 ml of a buffer solution containing 50 mM Tris-HCI, pH 7.5, 5 mM MgCI2, 5 mM 2-mereaptoethanol, 1 mM EDTA, and 20% glycerol (standard buffer). The suspension was centrifuged at 30,000 X g for 10 min and the supernatant solution at 30,000 rpm in the Spinco no. 30 rotor for 2 hr. To the high-speed supernatant solution were added (per ml) with mixing, 0.117 ml 20% (w/w) Dextran 500, 0.310 ml 30% (w/w) polyethylene glycol, and 0.33 gm NaCl. Mixing was continued for 1 hr, after which the suspension was centrifuged at 15,000 X g for 5 min. The supernatant solution was dialyzed against 4 liters of standard buffer solution until the conductivity of a 5 X 104 dilution was less than 1.3 ppm as NaCl (2-3 hr) and then adjusted to 35% saturation with solid ammonium sulfate. After being mixed for 10 min the suspension was centrifuged at 10,000 X g for 5 min and the lower phase was removed and adjusted to 55% saturation

Replication of RNA viruses. V. Template activity of synthetic ribopolymers in the Q-beta RNA polymerase reaction.

Investigation of the mechanism of viral RNA synthesis has been greatly facilitated by use of the RNA-dependent RNA polymerase isolated from cells infected with the single-stranded RNA phage Q,B. In the reaction catalyzed by this enzyme, Q,B RNA and ribonucleoside triphosphates are required. The RNA product has a molecular weight of approximately 1 X 106 daltons, corresponding to that of Q3 RNA, and may be produced in manyfold excess of the amount added to the reaction." 2 As part of our studies of the enzyme, we have investigated the initiation of RNA synthesis. In general, synthesis of polynucleotide chains is thought to involve a nucleophilic attack by the 3'-hydroxyl group of the polynucleotide on the a-phosphate of the entering nucleoside triphosphate.3-5 By this mechanism, newly synthesized RNA could contain a nucleoside triphosphate as one of the termini of the molecule. Maitra and Hurwitz5 and Bremer et al.6 have described the presence of such termini in the RNA product of the DNA-dependent RNA polymerase reaction. Methods.-y-P82-adenosine 5'-triphosphate (ATP), y-P32-guanosine 5'-triphosphate (GTP), and -y-P32-uridine triphosphate (UTP) were prepared by the exchange of P32 with the corresponding ribonucleoside triphosphate by the method of Penefsky et al.7 The reaction was allowed to proceed until all of the Pi32 was converted into a charcoal-adsorbable form. The Y-P32-nucleoside triphosphates were then purified by chromatography on Dowex-1-Cl-. y-P32-cytidine 5'-triphosphate (CTP) was prepared from y-P32-ATP and cytidine 5'-diphosphate (CDP) in the reaction catalyzed by nucleoside diphosphokinase8 and was purified by chromatography on Dowex-l

A. K. Banerjee

Papers

Synthesis of bacteriophage Q-beta RNA.

Replication of RNA viruses. V. Template activity of synthetic ribopolymers in the Q-beta RNA polymerase reaction.

Replication of RNA viruses, IV. Initiation of RNA synthesis by the q-beta RNA polymerase.