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BglG, the transcriptional antiterminator of the bgl system, interacts with the β′ subunit of the Escherichia coli rna polymerase

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第8期96卷 4336-4341页

作者： Nussbaum-Shochat, A Amster-Choder, O Hebrew Univ Jerusalem Hadassah Med Sch Dept Mol Biol IL-91120 Jerusalem Israel

The Escherichia coli BglG protein antiterminates transcription at two terminator sites within the bgl operon in response to the presence of P-glucosides in the growth medium. BglG was previously shown to be an rna-binding protein that recognizes a specific sequence located just upstream of each of the terminators and partially overlapping with them. We show here that BglG also binds to the E. coli rna polymerase, both in vivo and in vitro. By using several techniques, we identified the beta subunit of rna polymerase as the target for BglG binding. The region that contains the binding site for BglG was mapped to the N-terminal region of beta. The beta subunit, produced in excess, prevented BglG activity as a transcriptional antiterminator. Possible roles of the interaction between BglG and the polymerase beta subunit are discussed.

关键词： ATP结合匣式转运子细菌蛋白质类/分离和提纯细菌蛋白质类/代谢印迹法蛋白质载体蛋白质类/生物合成载体蛋白质类/分离和提纯载体蛋白质类/代谢色谱法亲和指导dna的rna聚合酶类/化学指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢大肠杆菌/代谢大肠杆菌蛋白质类大分子物质麦芽糖结合蛋白质类单糖转运蛋白质类质粒 rna结合蛋白质类/分离和提纯 rna结合蛋白质类/代谢重组融合蛋白质类/生物合成重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢序列缺失

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Functional analysis of two maize cdnas encoding T7-like rna polymerases

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PLANT CELL 1999年第5期11卷 911-926页

作者： Chang, CC Sheen, J Bligny, M Niwa, Y Lerbs-Mache, S Stern, DB Cornell Univ Boyce Thompson Inst Plant Res Ithaca NY 14853 USA Harvard Univ Sch Med Dept Genet Boston MA 02114 USA Massachusetts Gen Hosp Dept Biol Mol Boston MA 02114 USA Univ Grenoble 1 Lab Genet Mol Plantes F-38041 Grenoble 9 France Univ Shizuoka Sch Food & Nutrit Sci Shizuoka 422 Japan

We have characterized two maize cdnas, rpoTm and rpoTp, that encode putative T7-like rna polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of similar to 100 kD. Their presence was correlated with rna polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid rna polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

关键词：氨基酸序列细胞区室化叶绿体/酶学克隆分子保守序列 dna 互补/遗传学指导dna的rna聚合酶类/遗传学指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢基因剂量绿色荧光蛋白质类发光蛋白质类/遗传学发光蛋白质类/分离和提纯线粒体/酶学分子序列数据 rna 信使/生物合成 rna 信使/遗传学 rna 植物/生物合成 rna 植物/遗传学重组融合蛋白质类/分离和提纯序列同源性氨基酸组织分布病毒蛋白质类玉米/酶学玉米/遗传学

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Histidine codons appended to the gene encoding the RP022 subunit of vaccinia virus rna polymerase facilitate the isolation and purification of functional enzyme and associated proteins from virus-infected cells

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VIROLOGY 1999年第2期258卷 469-479页

作者： Katsafanas, GC Moss, B NIAID Viral Dis Lab NIH Bethesda MD 20892 USA

Vaccinia virus encodes a eukaryotic-like rna polymerase composed of two large and six small subunit protein species. A replication-competent virus with 10 histidine codons added to the single endogenous J4R open reading frame was constructed. The altered migration of the 22-kDa subunit of rna polymerase on SDS-polyacrylamide gel electrophoresis confirmed that J4R encoded the RPO22 subunit and that the mutant virus was genetically stable. The histidine-tagged rna polymerase bound quantitatively to metal-affinity resins and was eluted in an active form upon addition of imidazole. Glycerol gradient sedimentation of the eluted fraction indicated that most of the RPO22 in infected cells is associated with rna polymerase. Using stringent washing conditions, metal-affinity chromatography resulted in a several hundred-fold increase in rna-polymerase-specific activity, and substantially pure enzyme was obtained with an additional conventional chromatography step. When mild conditions were used for washing the metal-affinity resin, the vaccinia virus-encoded capping enzyme, early transcription factor, and nucleoside triphosphate phosphohydrolase I specifically co-eluted with the tagged rna polymerase, consistent with their physical association. The ability to selectively bind rna polymerase to an affinity column provided a simple and rapid method of concentrating and purifying active enzyme and protein complexes.

关键词：细胞系长尾猴黑色谱法亲和密码子指导dna的rna聚合酶类/遗传学指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢基因病毒 HeLa细胞组氨酸金属重组遗传痘苗病毒/酶学痘苗病毒/遗传学病毒蛋白质类/遗传学病毒蛋白质类/分离和提纯病毒蛋白质类/代谢动物人类

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FRACTIONATION OF rna-POLYMERASE SUBSPECIES FROM SOYBEAN HYPOCOTYLS BY ISOELECTRIC-FOCUSING IN SEPHADEX

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ANALYTICAL BIOCHEMISTRY 1976年第1期76卷 311-320页

作者： JOHNSON, LK LIEBKE, HH OBRIEN, TJ UNIV SO CALIF ALLAN HANCOCK FDN DEPT BIOL CHEM LOS ANGELES CA 90007 USA

rna polymerase enzymes isolated from chromatin of 6 day old soybean hypocotyls were resolved into 2 major and 1 minor species of activity by DEAE-Sephadex chromatography. A soluble form of the enzyme, isolated from the postchromatin supernatant fraction, shows a broad peak of activity when fractionated by this method. The elution characteristics and .alpha.-amanitin sensitivity data indicate the 2 major chromatin-bound activities are Class I and III enzymes, while the minor chromatin-bound activity and the soluble enzyme are representative of the Class II enzymes. Fractionation of these enzyme preparations by the new method of isoelectric focusing in Sephadex G-15 yields 5 distinct chromatin-bound and 4 soluble subspecies. The relationships of these observed activities to their parent DEAE classes are investigated, showing 2 subspecies within the Class I and III rna polymerase enzymes, respectively, and 4 subspecies within the Class II enzymes.

关键词：染色质/酶学指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢右旋糖酐类等电聚焦/方法同工酶类/分离和提纯同工酶类/代谢植物/酶学黄豆

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TRANSCRIPTION INVITRO OF EHRLICH ASCITES TUMOR dna AND CHROMATIN BY PURIFIED HOMOLOGOUS rna POLYMERASE-2 (OR POLYMERASE-B)

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1976年第2期173卷 564-576页

作者： CHAN, JYH LOOR, RM WANG, TY SUNY BUFFALO DIV CELL & MOLEC BIOL BUFFALO NY 14214 USA

An electrophoretically homogeneous rna polymerase II (α-amanitin sensitive) has been purified from Ehrlich ascites tumor cells and used to study transcription of homologous dna and chromatin. The general characteristics in the transcription of dna and chromatin by the purified rna polymerase are compared with respect to metal ion requirements and sensitivity toward spermine, rose bengal, and heparin. The fidelity of the tumor rna polymerase in transcription is examined by comparison with that of Micrococcus luteus rna polymerase. Only a limited portion of dna and chromatin is transcribed by the tumor rna polymerase, while the bacterial enzyme transcribes both templates extensively. By double-saturation of dna or chromatin with the two enzymes as well as kinetic studies, it is shown that the tumor rna polymerase, in contrast to the M. luteus rna polymerase, transcribes selective dna sequences.

关键词：癌 Ehrlich瘤/酶学癌 Ehrlich瘤/代谢染色质/代谢 dna 肿瘤/代谢指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢酶激活/药物作用肝素/药理学动力学镁/药理学锰/药理学微球菌属/代谢容积渗克分子浓度玫瑰红/药理学氯化钠/药理学精胺/药理学转录遗传动物男(雄)性小鼠

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INCREASES IN ACTIVITY OF SOLUBILIZED RAT-LIVER NUCLEAR-rna POLYMERASES FOLLOWING PARTIAL-HEPATECTOMY

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1975年第2期170卷 485-491页

作者： ORGANTINI, JE JOSEPH, CR FARBER, JL TEMPLE UNIV SCH MED FELS RES INST PHILADELPHIA PA 19140 USA TEMPLE UNIV SCH MED DEPT PATHOL PHILADELPHIA PA 19140 USA

Nuclei isolated 18 h following partial hepatectomy in the rat synthesize rna in vitro at twice the rate of liver nuclei from sham operated animals. This increased rna synthesis is accompanied by an increase in the α amanitin insensitive proportion of the total activity. Twice as much rna polymerase activity was solubilized from nuclei from hepatectomized animals as from sham operated animals. Fractionation of this solubilized rna polymerase activity on DEAE Sephadex showed increases in the activity of both polymerase I and polymerase II, with a proportionally greater increase in polymerase I. Such an increase in rna polymerase activity independent of the endogenous chromatin template was also demonstrated by inhibiting the function of the chromatin template in whole nuclei with actinomycin D and then assaying the polymerase activity with poly[d(A-T)]. It is concluded that increases in the activity of the rna polymerases themselves can account entirely for the changes in the rates of rna synthesis 18 h following partial hepatectomy.

关键词：细胞核/药物作用细胞核/酶学指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢更生霉素/药理学肝切除术肝/酶学肝再生/药物作用聚核苷酸类/药理学溶解度转录遗传/药物作用动物女(雌)性大鼠

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TRANSLATIONAL MAPPING OF BACTERIOPHAGE T7 rnaS SYNTHESIZED INVITRO BY PURIFIED T7 rna-POLYMERASE

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JOUrnaL OF MOLECULAR BIOLOGY 1975年第1期98卷 57-67页

作者： NILES, EG CONDIT, RC YALE UNIV DEPT THERAPEUTIC RADIOLNEW HAVENCT 06510 YALE UNIV DEPT MOLEC BIOPHYS & BIOCHEMNEW HAVENCT 06510

Late bacteriophage T7 rna, synthesized in vitro from wild-type, amber and deletion mutant phage dnas using purified T7 rna polymerase, was used to program an rna-dependent in vitro protein synthesizing system. Products were analyzed by electrophoresis on polyacrylamide gels. When unfractionated late T7 rna was translated, almost all late T7 proteins were synthesized, demonstrating that the entire late region of T7 dna is transcribed by T7 rna polymerase in vitro and that the leftmost initiation site for T7 rna polymerase lies between the left ends of genes 1 and 1.3 . Translation of fractionated species of in vitro synthesized late T7 rna, purified by polyacrylamide gel electrophoresis, showed that T7 rna species IV and V stimulate the synthesis of the T7 genes 9 and 10 proteins and of the gene 10 protein, respectively. Species VI does not encode any T7 proteins. The major products of translation of species III are genes 9, 10 and 17 polypeptides. These results demonstrate the existence of promoters for T7 rna polymerase between genes 8 and 9 and between genes 9 and 10 , and a termination site between genes 10 and 11 . They are also consistent with the finding (Golomb & Chamberlin, 1974 b ) that rna species IIIb and VI are transcribed from the extreme right end of T7 dna.

关键词：染色体图大肠杆菌噬菌体/代谢指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢大肠杆菌/代谢遗传密码基因型突变蛋白质生物合成 rna 病毒/代谢种特异性

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TRANSCRIPTION OF CHROMATIN BY AN rna-POLYMERASE OF CALF THYMUS WHICH IS SENSITIVE TO HIGH-CONCENTRATIONS OF ALPHA-AMANITIN

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 1975年第2期169卷 545-554页

作者： KESHGEGIAN, AA ACKERMAN, S FURTH, JJ UNIV PENN SCH MEDDEPT BIOCHEMPHILADELPHIAPA 19174 UNIV PENN SCH MEDDEPT MICROBIOLPHILADELPHIAPA 19174 UNIV PENN SCH MEDDEPT PATHOLPHILADELPHIAPA 19174

rna polymerase which is sensitive to high concentrations of α-amanitin has been obtained by low-salt extraction of whole calf thymus tissue. Some characteristics of this Form III-type polymerase have been compared with Form II. Both enzymes require a higher concentration of purine nucleoside triphosphates compared to pyrimidine nucleoside triphosphates. The Form III-type polymerase is larger than Form II. It transcribes native dna better than denatured dna and transcribes chromatin efficiently. Under various salt and metal conditions Form II transcribes native dna and chromatin poorly relative to denatured dna. These results suggest that Form III rna polymerase is a useful enzyme with which to study the transcription of chromatin in vitro and may possess some factor (which Form II lacks) which is necessary for transcription of double-stranded templates.

关键词：鹅膏菌素类/药理学硫酸铵/药理学离心法梯密度染色质/药物作用染色质/代谢色谱法离子交换指导dna的rna聚合酶类/分离和提纯指导dna的rna聚合酶类/代谢酶激活/药物作用同工酶类/分离和提纯同工酶类/代谢动力学镁/药理学锰/药理学氯化钾/药理学模板遗传胸腺/药物作用胸腺/酶学时间因素转录遗传/药物作用动物牛

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TRANSCRIPTION OF dna AND CHROMATIN WITH CALF THYMUS rna POLYMERASE-B INVITRO

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JOUrnaL OF MOLECULAR BIOLOGY 1975年第2期95卷 257-269页

作者： CEDAR, H HEBREW UNIV HADASSAH MED SCHDEPT BIOCHEMJERUSALEMISRAEL

The interaction of calf thymus rna polymerase B with dna and chromatin has been studied under conditions which permit direct measurement of binding sites capable of supporting chain initiation. The number of such sites is determined by counting the number of growing rna chains, using either sucrose gradient ultracentrifugation or incorporation of [γ- 32 P]ATP and [γ 32 P]GTP. These measurements show that there is one site per 40,000 nucleotide pairs on dna and one-tenth that number on chromatin. This is in contrast to the bacterial rna polymerase, which has many more initiation sites both on dna and on chromatin.

关键词：腺苷三磷酸结合部位离心法梯密度染色质 dna dna 单链指导dna的rna聚合酶类/分离和提纯大肠杆菌/酶学鸟苷三磷酸动力学模板遗传胸腺/酶学转录遗传动物牛

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