Primer-independent abortive initiation by wheat-germ RNA polymerase B (II)

Highly purified RNA polymerase B (II) from wheat germ catalyses the formation of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires bivalent cations such as Mn2+ or Mg2+ and proceeds linearly for several hours. It is strongly inhibited by 1 microgram/ml alpha-amanitin or 2 micrograms/ml heparin. The reaction strictly depends on the addition of a specific linear or circular DNA template, such as the plasmid pSmaF or a DNA fragment containing the gene for nopaline dehydrogenase. Bacteriophage T7 D111 DNA has almost no template activity. The start sites for dinucleotide synthesis on the template are limited. With the DNA fragment containing the gene for nopaline dehydrogenase only pppApA and pppApU are synthesised substantially whereas pppUpU is formed only in trace amounts. No significant dinucleotide synthesis is observed with other ribonucleoside triphosphates either singly or in a combination of two. The various regions of the DNA fragment differ distinctly in template activity.     

Identification of host-derived subunits of phage SP RNA-dependent RNA polymerase (SP replicase).

Phage SP RNA-dependent RNA polymerase (SP replicase) was purified from Escherichia coli infected with RNA phage SP. The enzyme was found to be composed of four non-identical polypeptides, i.e. subunits I, II, III, and IV and molecular weights of 74,000, 69,000, 47,000, and 36,000 daltons, respectively. As in the case of phage Qbeta replicase, the largest polypeptide is identical with the ribosomal protein S1, and subunits III and IV with polypeptide chain elongation factors EF-Tu and EF-ts, respectively.. This is based on the behaviour of the subunits on SDS-polyacrylamide gel electrophoresis, isoelectric focusing and immunological cross-reaction. Subunits I, III, and IV of SP replicase are derived from the host cell, while subunit II is coded by phage RNA genome. The striking coincidence of the composition and entity of the structural components of SP replicase with those of Qbeta replicase may indicate the structural and functional requirements of host-derived polypeptides in RNA replicase. The binding activity of S1 (in 70S ribosome comples) to poly (U) is retained in SP replicase complex. In contrast, the GDP binding activity of EF-Tu is masked in SP replicase. It is concluded that S1 is required functionally whereas EF-Tu.EF-Ts are required structurally in RNA replicase.     

Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template

RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3'-end. We have previously shown that NS5B can utilize the 3'-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3'-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3'-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3'-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3'-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3'-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3'-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3'-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells.     

SP6 RNA polymerase efficiently synthesizes RNA from short double-stranded DNA templates.

SP6 DNA-dependent RNA polymerase, like T7 RNA polymerase, can be used to synthesize RNA sequences from short DNA templates which contain the 18 base pair promoter region. Use of SP6 polymerase extends the range of possible 5' sequences of RNA products, since the preferred SP6 start site (of the RNA product) is 5'GAAGA, while T7 polymerase prefers 5'GGGAG. The SP6 start site can be advantageous in large-scale syntheses where high concentrations of RNA can lead to aggregation. Using the limited number of DNA templates described here, there appears to be a significant difference between the two enzymes: SP6 polymerase requires a complete duplex DNA substrate for efficient synthesis, unlike the T7 enzyme which works efficiently when only the 18 base promoter region is double-stranded. SP6 polymerase consistently produces higher yields of RNA than does T7 polymerase, and the reactions can be easily scaled up to produce milligram quantities of RNA.