The RNA Polymerase Structure–Function Study (1962–2001)

The study of RNA polymerase initiated by R.B. Khesin has been conducted for about forty years at the laboratory founded by him (since 1989, in collaboration with A. Goldfarb's laboratory). Genetic methods are used in combination with methods of the specific chemical crosslinks of nucleic acids with proteins. The paper assesses the main results of the study in comparison with the recent high-resolution X-ray crystallographic data. A short comparative summary of the RNA polymerase structure is presented for bacteria, archaebacteria, and eukaryotic organelles and nuclei. A brief history of the RNA polymerase study is also given.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

Alpha-amanitin blocks translocation by human RNA polymerase II

Our laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using alpha-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with alpha-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, alpha-amanitin inhibits elongation at each translocation step.     

Processivity of ribozyme-catalyzed RNA polymerization

The "RNA world" hypothesis proposes that early in the evolution of life, before the appearance of DNA or protein, RNA was responsible both for encoding genetic information and for catalyzing biochemical reactions. Ribo-organisms living in the RNA world would have replicated their RNA genomes by using an RNA polymerase ribozyme. Efforts to provide experimental support for the RNA world hypothesis have focused on producing such a polymerase, and in vitro evolution methods have led to the isolation of a polymerase ribozyme that catalyzes primer extension which is accurate and general, but slow. To understand the reaction of this ribozyme, we developed a method of measuring polymerase processivity that is particularly useful in the case of an inefficient polymerase. This method allowed us to demonstrate that the polymerase ribozyme, despite its inefficiency, is partially processive. It is currently limited by a low affinity for the primer-template duplex, but once it successfully binds the primer-template duplex in the productive alignment, it catalyzes an extension reaction that is so rapid that it can occur multiple times during the short span of a single binding event. This finding contributes to the understanding of one of the more sophisticated activities yet to be generated de novo in the laboratory and sheds light on the parameters to be targeted for further optimization.     

COMPARISON AND CHARACTERIZATION OF NUCLEAR ISOLATION PROCEDURES AS APPLIED TO CHICK OVIDUCT

A number of methods for the preparation of chick oviduct nuclei have been compared. Nuclei have been isolated in hypertonic sucrose and citric acid and the product has been characterized with respect to cleanliness, ultrastructure, RNA polymerase activity, RNA integrity, and chromatin composition. The study demonstrates that the choice of oviduct nuclear isolation procedure will depend markedly on the purpose for which the nuclei are required. Thus, nuclei prepared entirely in high-molarity sucrose retain the highest levels of RNA polymerase. Those prepared rapidly in the presence of citric acid retain nuclear RNA in an essentially undegraded state. Finally, a bulk preparation is described which, because of its adaptability and high yield of morphologically intact nuclei using large amounts of tissue, is ideal for use in preparing chromatin. Conditions are described by which isolated nuclei can be stored for up to 6 months and retain their morphology, chemical characteristics, and RNA polymerase activity.     

RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.

20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.     

Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy

A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly[d(A-T)].poly[d(A-T)] and poly[d-(G-C)].poly[d(G-C)]. As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature. An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C. A decrease was observed in the case of poly[d(A-T)].poly[d(A-T)] at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly[d(G-C)].poly[d(G-C)]. Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C. Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin. The interaction between pyrenyl RNA polymerase and poly[d-(A-T)].poly[d(A-T)] at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

3'-end labeling of RNA with recombinant yeast poly(A) polymerase.

Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.     

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.     

DNA-Dependent RNA Polymerases from Artemia salina

Embryos and larvae of the brine shrimp, Artemia salina , provide a useful biological system for biochemical studies of animal development. Dormant encysted embryos can be cultured readily in the laboratory to provide large quantities of free-swimming nauplius larvae. The rate of synthesis of all classes of RNA in swimming larvae declines markedly between 24 and 72 h after immersion of dormant embryos in sea water. Nuclei were isolated from 24–72 h larvae and RNA polymerase activity was measured under conditions in which the nuclei remained intact. Total RNA polymerase activity of isolated nuclei decreased in parallel with RNA synthesis in vivo. RNA polymerases were solubilized from nuclei and fractionated by chromatography on DEAE-cellulose. The levels of both RNA polymerases I and II also decreased in parallel with RNA synthesis in vivo. The specific activity of highly purified RNA polymerase II was determined by comparison of enzyme activity with the mass of RNA polymerase II subunits displayed on SDS gels. The specific activities of RNA polymerase II preparations from 24 and 72 h larvae were identical. The number of polymerase II molecules was estimated from the mass of the subunits. The number of molecules per nucleus declined from 20,000 at 24 h to 3500 at 72 h.     

Cloning, expression and characterisation of a Family B ATP-dependent phosphofructokinase activity from the hyperthermophilic crenarachaeon Aeropyrum pernix

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.