The subunit structures of soluble and chromatin-bound RNA polymerase II from soybean

DNA-dependent RNA polymerase II is present in two forms, IIa and IIb, in germinating soybean. Form IIa is the dominant form of the enzyme in ungerminated embryos and appears to be a soluble enzyme. Form IIb increases in amount as germination progresses and is tightly bound to the chromatin template. The subunit structures of soybean RNA polymerases IIa and IIb are identical except for the molecular weights of their largest subunits which are 200,000 daltons and 170,000 daltons for IIa and IIb, respectively. The enzymes have seven common subunits: 142,000, 42,000, 26,000, 20,000, 16,000, 15,000, and 14,000 daltons.     

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.     

RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.     

A soluble biotinyl-alpha-amanitin affinity probe for the isolation of RNA polymerase B

Utilizing the 6'-hydroxyindole moiety of alpha-amanitin for substitution, biotinyl-alpha-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-alpha-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 X 10(-8) M as compared with a Ki of 5 X 10(-9) M for natural alpha-amanitin. RNA polymerase B complexed with biotinyl-alpha-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the anatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-alpha-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.     

Effects of abscisic acid on the soluble RNA polymerase activity in maize coleoptiles

Summary Effects of abscisic acid (ABA) on the polymerase activity have been investigated. The specific activity of the RNA polymerase enzyme decreased when coleoptiles were preincubated for 6 hours or longer in 3.8×10^-5M ABA. Inhibition of RNA synthesis could however already be detected after 3 hours ABA treatment.Inclusion of (RS) cis-trans ABA in the grinding medium decreased the polymerase activity; inclusion of (RS) trans-trans ABA in the medium only had a small effect on the activity. Addition of (RS) cis-trans and (RS) trans-trans ABA to the RNA polymerase assay system also gave a slight inhibition of activity.The strong inhibition when (RS) cis-trans ABA was included in the grinding medium indicates that the hormone interacts with an activator molecule which is not present in the partly purified RNA polymerase solution.It is suggested that ABA may have more than one mode of action.     

Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.     

Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.     

Competition of rifampicin with binding of substrate and RNA to RNA polymerase

The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A1 promoter of the mutant D111 of bacteriophage T7 is distinctly and specifically reduced not only by the third template-directed nucleotide, CTP, but also by CMP. The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin. The synthesis of pppApU is also strongly reduced after preincubation of the enzyme with RNA. This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA. On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly alpha 2 beta.     

RNA polymerase pushing

Molecular motors can exhibit Brownian ratchet or power stroke mechanisms. These mechanistic categories are related to transition state position: An early transition state suggests that chemical energy is stored and then released during the step (stroke) while a late transition state suggests that the release of chemical energy rectifies thermally activated motion that has already occurred (ratchet). Cellular RNA polymerases are thought to be ratchets that can push each other forward to reduce pausing during elongation. Here, by constructing a two-dimensional energy landscape from the individual landscapes of active and backtracked enzymes, we identify a new pushing mechanism which is the result of a saddle trajectory that arises in the two-dimensional energy landscape of interacting enzymes. We show that this mechanism is more effective with an early transition state suggesting that interacting RNAPs might translocate via a power stroke.