Biomedical subjects

D Job

Publications and source records attributed to D Job.

At least 73 records · Page 4Linked to original sources

PubMed1994

Effect of disulfide and sulfhydryl reagents on abortive and productive elongation catalyzed by Escherichia coli RNA polymerase.

The effect of disulfide and sulfhydryl reagents on the rate of abortive and productive elongation has been studied using Escherichia coli RNA polymerase holoenzyme and poly[d(A-T)] as template. In the presence of UTP as a single substrate and UpA as a primer, the enzyme catalyzed efficiently the synthesis of the trinucleotide product UpApU. Incubation of RNA polymerase with 1 mM 2-mercaptoethanol resulted in a 5-fold increase of the rate of UpApU synthesis. In contrast, incubation of the enzyme with 1 mM 5,5'-dithio-bis(2-nitrobenzoic) acid resulted in a 6-fold decrease of the rate of abortive elongation. Determination of the steady state kinetic constants associated with UpApU synthesis disclosed that the disulfide and sulfhydryl reagents mainly affected the rate of UpApU release from the ternary transcription complexes and therefore influenced the stability of such complexes.

DNA-Directed RNA Polymerases↗

PubMed1993

Spectrum of DNA--platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases.

Double-stranded DNA oligomers were constructed to evaluate the effect of bifunctional and monofunctional platinum(II) complexes at the level of DNA transcription. They contained a single lesion, which is either a cis-[Pt(NH3)2(d(GpTpG))] intrastrand cross-link, a trans-[Pt(NH3)2(d(GpTpG))] intrastrand cross-link, a cis-[Pt(NH3)2(d(GpC/GpC))] interstrand cross-link, or a (diethylenetriamine)-platinum(II)-dG adduct. The synthetic duplexes were multimerized and then used as templates in dinucleotide-primed reactions catalyzed by prokaryotic or eukaryotic RNA polymerases. Reactions were conducted in the presence of a single triphosphate substrate (single-step addition reaction) or of a combination of triphosphate substrates, permitting elongation of the trinucleotide products to longer RNA chains (productive elongation reaction), respectively. In transcription of the platinated strands, none of the DNA adducts provided an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. However, the single-step addition reactions were much more impeded from transcription of bifunctional adduct-containing templates as compared to those containing monofunctional lesions. Productive elongation was irreversibly blocked in transcription of the platinated strand of templates containing a cis-d(G*pTpG*) intrastrand cross-link or a cis-d(G*pC/G*pC) interstrand cross-link. In both cases transcription stopped at the level of the lesion. Termination occurred also several nucleotides before the elongation complexes reached the interstrand cross-link. A substantial amount of the RNA polymerase molecules was able of bypassing the trans-d(G*pTpG*) cross-links. In all the cases single-step addition reactions were enhanced on the template strand complementary to that containing the intrastrand cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?

A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5'-phosphate cofactor to be identified. The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution. The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor. The comparison also suggests that the 5'-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme.

Amino Acid Sequence↗

PubMed1992

Isolation and kinetic properties of acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) chloroplasts overexpressed in Escherichia coli.

Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids. Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria. To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme. A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase. Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium. Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate). Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site. The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses. Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light. As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.

2-Acetolactate Mutase↗

PubMed1992

Purification of assembly-competent tubulin from Saccharomyces cerevisiae.

We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.

Animals↗

PubMed1992

Ca(2+)-calmodulin regulated effectors of microtubule stability in neuronal tissues.

In general, microtubules are labile structures which depolymerize at low temperature and are sensitive to Ca2+. However, in brain tissue, axonal microtubules are disassembly-resistant and can exist without attachment to a microtubule organizing center. Stable microtubules cannot be purified by usual recycling procedures and this has made the elucidation of the molecular mechanisms involved in their stabilization difficult. This paper summarizes previous work in our laboratories, aimed at the identification of brain microtubule stabilizing proteins. We present assay methods which allow the detection of microtubule stability effectors in complex extracts and in chromatographic column fractions. Applied to brain crude extracts, they result in the isolation of Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated proteins. One, called STOP, appears to account for microtubule stabilization in neurons. A second protein with similar activity is myelin basic protein. Non-neuronal tissues also contain Ca(2+)-calmodulin-regulated effectors which appear to differ in structure from their neuronal counterparts. Thus, in all tissues examined, microtubule stability seems to be accounted for by unique Ca(2+)-calmodulin regulated proteins, showing tissue specificity.

Animals↗

PubMed1992

Stabilization of sea urchin flagellar microtubules by histone H1.

Complex microtubule assemblies are essential components of eukaryotic cilia and flagella. They are extremely stable and are not affected by agents that normally induce polymer disassembly. The molecular basis of this microtubular stability is unknown, and it is not related to any feature of the constitutive tubulin. In sea urchin sperm flagella, axonemal microtubules are found to be stabilized by a protein identical to histone H1, a result that defines a new role for this histone and provides evidence for a concerted evolution of chromatin and microtubular structures.

Amino Acid Sequence↗

PubMed1992

Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Gravitational symmetry breaking in microtubular dissipative structures.

Reduction-diffusion theories can account for both morphogenesis and the sensitivity of biological systems to weak fields. They predict that gravity can cause the symmetry breaking that is necessary for pattern formation. Microtubules play an important role in organizing the cell, and recent studies hae shown that they can form in vitro dissipative structures. We have found that these structures show patterns of microtubular orientation that are gravity dependent and that the gravitational field causes symmetry breaking. This behavior, which cannot be explained by convection, is in accordance with the theory of dissipative structures. These results suggest that microtubular dissipative structures may play an important role both in morphogenesis and in accounting for the sensitivity of biological systems to weak fields. They aso provide another explanation for biological gravitropism.

Animals↗

PubMed1992

A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin alpha-amanitin.

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.

Amanitins↗

PubMed1992

Accuracy of wheat-germ RNA polymerase II. General enzymatic properties and effect of template conformational transition from right-handed B-DNA to left-handed Z-DNA.

We investigated the accuracy of the insertion process in RNA chain elongation catalyzed by wheat germ RNA polymerase II. Error frequencies varied from 1 misinserted nucleotide per 250 polymerized correct substrates to less than 1 in 2 x 10(5), depending on template sequence and nature of the divalent metal cofactor. Higher error ratios were observed in the presence of Mn2+ compared to Mg2+, and with alternating poly[d(G-C)].poly[d(G-C)] compared to poly[d(A-T)].poly[d(A-T)]. In this latter case the eukaryotic RNA polymerase was as accurate as Escherichia coli RNA polymerase holo-enzyme. The fidelity of wheat germ RNA polymerase II was also examined in transcription of polynucleotide templates in the poly[d(G-C)] family adopting either the right-handed B or left-handed Z conformations. Error ratios for noncomplementary ATP increased markedly under experimental conditions favoring the B-to-Z conformational transition of the alternating copolymers. In accordance with the results of previous studies, the rate of productive elongation, i.e. the synthesis of poly[r(G-C)], was depressed, suggesting that the decreased accuracy of the enzyme derived from an altered competence of the enzyme to form elongation complexes on the left-handed DNA. As judged by the large difference in apparent Km values of the enzyme for complementary and noncomplementary nucleoside triphosphates, part of the discrimination between substrates seemed to take place at the initial binding step. Furthermore, the results indicate that wheat germ RNA polymerase II was able to elongate a primer with a 3'-terminal mismatch, and thus to incorporate the mismatched nucleotide stably in the nascent RAN. However, the probability of productive RNA chain elongation was much lower with noncognate than with the complementary substrates.

Adenosine Triphosphate↗

PubMed1992

RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II).

Two duplexes (20-mers) were constructed containing either a single cis-[Pt(NH3)2[d(GpG)]] or cis-[Pt(NH3)2[d(ApG)]] intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II). These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases. Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates. Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites. In particular, binding affinity between E. coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent Km of enzyme for the platinated polymer is increased by a factor of 4-5. In contrast, binding affinity between the RNA polymerase and the d(ApG)-containing template is not affected by modification of the d(ApG) site by cis-diamminedichloroplatinum(II). Similar experiments were carried out with synthetic templates containing the adducts at the d(GpG) sites, in which one of the two platinated dG residues is paired with a dT residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types.

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.

Animals↗

PubMed1991

Characterization of a major brain tubulin variant which cannot be tyrosinated.

Brain tubulin preparations contain an abundant type of tubulin which does not undergo the normal cycle of tyrosination-detyrosination, and whose nature is still unknown. We have used peptide sequence analysis and mass spectrometry combined with immunological procedures to show that this non-tyrosinatable tubulin has a specific primary structure. It differs from the tyrosinated isotype in that it lacks a carboxy-terminal glutamyl-tyrosine group on its alpha-subunit. Thus, non-tyrosinatable tubulin originates from a well-defined posttranslational modification of the tubulin primary structure which is located at the expected site of activity of tubulin tyrosine ligase. This probably accounts for the reason why it cannot be tyrosinated. The significance of this abundant brain isotubulin and the metabolic pathway involved in its formation remain to be elucidated. This should shed light on the relation between the structural diversity of the carboxy terminus of alpha-tubulin and the regulation of functional properties of microtubules.

Amino Acid Sequence↗

PubMed1991

Transcription of synthetic DNA containing sequences with dyad symmetry by wheat-germ RNA polymerase II. Increased rates of product release in single-step addition reactions.

Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat-germ RNA polymerase II and a series of double-stranded template polymers containing palindromic repeating motifs of 6-16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC].d[TAT(GC)nG], with n = 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double-stranded methylated polymers, containing the repeating units d(ATAm5CGm5C).d(TATGm5CG) and d[ATA(m5CG)6m5C].d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single-step addition of CTP to a CpG primer catalysed by wheat-germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single-step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G-C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12-fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G-C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA-primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II.

Base Sequence↗

PubMed1991

Transcription by eucaryotic and procaryotic RNA polymerases of DNA modified at a d(GG) or a d(AG) site by the antitumor drug cis-diamminedichloroplatinum(II).

We have investigated whether DNA modified at a d(GG) or a d(AG) site by the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP) can be used as template by wheat germ RNA polymerase II. The templates used in the present study were obtained by ligation of double-helical oligodeoxyribonucleotides, containing 18 pyrimidine bases and 2 central dG, or dA and dG, bases on one strand and 18 purine bases and 2 central dC, or dT and dC, bases on the complementary strand. Therefore, the cis-DDP adducts are only present on one strand of each of the two templates and are regularly spaced by 18 pyrimidine bases. These constructs allowed us to investigate the effect of cis-DDP on transcription of the platinated strand and of the complementary unplatinated sequence. Transcription experiments were carried out in the presence of dinucleotide primers and either a single triphosphate substrate (abortive elongation) or the full set of triphosphate substrates dictated by the template sequence (productive elongation). The results show that the eucaryotic RNA polymerase can catalyze dinucleotide-primed reactions on platinated DNA. However, the eucaryotic enzyme behaved very differently depending on which strand was transcribed. Thus, transcription elongation was completely blocked on the strand carrying the metal complex, whereas transcription elongation was not blocked on the complementary template strand. However, on this latter strand and with the platinated polymers, productive elongation was slightly inhibited. Furthermore, abortive elongation leading to dinucleotide-primed trinucleotide formation was enhanced on the template strand complementary to that carrying the cis-DDP adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed1991

The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus.

Centrosomes isolated from various sources, including human cells, have the capacity to induce parthenogenetic development when injected into unfertilized amphibian eggs. We recently isolated calf thymus centrosomes and showed that they differ structurally and functionally from previously isolated centrosomes of KE37 cells, in that the two centrioles in calf thymocytes are linearly associated by their proximal ends through a mass of electron dense material and nucleate few microtubules from their distal ends (Komesli, S., F. Tournier, M. Paintrand, R. Margolis, D. Job, and M. Bornens. 1989. J. Cell Biol. 109:2869-2878). We report here that these centrosomes are also unable to induce egg cleavage and examine the various possibilities which could account for this lack of competence. The results show that: (a) the kinetics of microtubule assembly on calf thymus centrosomes in Xenopus extracts are comparable to those of KE37 centrosomes; (b) centrosomes isolated from thymus of calves raised under controlled conditions (without anabolic agents) also lack competence; (c) centrosomes isolated from bovine cells of other tissues are competent; (d) centrosomes isolated from thymus of three other species (rat, mouse, and human) are competent. Since the lack of activity of calf thymus centrosomes apparently was not linked to species or tissue differences, we compared the ultrastructure of the centrosomes in the various centrosome preparations. The results show a strict correlation between the linear arrangement of centrioles and the lack of activity of the centrosomes. They suggest that the centrosome cycle can be blocked when the centrioles are prevented from separating into a nonlinear configuration, a step which might be critical for the initiation of procentriole budding. They also indicate that the centrosome may be involved in the G0-G1 transition.

Animals↗

PubMed1990

Analysis of wheat-germ RNA polymerase II by trypsin cleavage. The integrity of the two largest subunits of the enzyme is not mandatory for basal transcriptional activity.

When wheat-germ RNA polymerase II is subjected to mild proteolytic attack in the presence of trypsin, the resulting form of the enzyme migrates as a single species on electrophoresis in native polyacrylamide gels, with an apparent Mr significantly smaller than that of the native enzyme. Analysis by denaturing gel electrophoresis of the truncated eukaryotic polymerase revealed that the two largest subunits of the native enzyme, i.e. the 220,000-Mr and 140,000-Mr subunits, were cleaved, giving rise to shorter polypeptide chains of Mr 172,800, 155,000, 143,000, 133,800, 125,000 and 115,000. The use of affinity-purified antibodies directed against each of the two large subunits of the native enzyme allowed us to probe for possible precursor/product relationships between the 220,000-Mr and 140,000-Mr subunits of wheat-germ RNA polymerase II and their breakdown products generated in the presence of trypsin. None of the smaller subunits of the plant RNA polymerase II appeared to be sensitive to trypsin attack. The results indicate that the truncated RNA polymerase retained a multimeric structure, and therefore that the proteolyzed largest subunits of the enzyme remained associated with the smaller ones. Furthermore, in transcription of a poly[d(A-T)] template, the catalytic activity of the proteolyzed form of wheat-germ RNA polymerase II was identical to that of the native enzyme. Therefore, the protein domains that can be deleted by the action of trypsin from the two large subunits of the plant transcriptase are not involved in DNA binding and/or nucleotide binding, and do not play an important role in template-directed catalysis of phosphodiester bond formation.

Electrophoresis, Polyacrylamide Gel↗